Sign up to receive free email alerts when patent applications with chosen keywords are publishedSIGN UP

Abstract:

Disclosed are water soluble polymeric conjugates comprising the structure
POLY-[Y--S--S-A]x, where POLY is a water soluble polymer; Y is a
hydrocarbon-based spacer group, x is 1 to 25, S--S is a disulfide group
attached to an sp3 hybridized carbon of Y; and A is a covalently
linked residue of a pharmacologically active molecule. Preferably, the
water soluble polymer is a PEG polymer. Also disclosed are polymeric
reagents useful to prepare such conjugates, and methods of their
formation and use.

Claims:

1. A polymer conjugate comprising the structure: POLY-[Y--S--S-A]x
wherein: POLY is a water soluble polymer; Y has the formula
--(CR1R2)n--, where n is 4 to 8, each of R1 and
R2 is independently selected from hydrogen, lower alkyl, lower
alkenyl, and a non-interfering substituent, where two groups R1 and
R2 on different carbon atoms may be linked to form a cycloalkyl or
aryl group; S--S is a disulfide group attached to an sp3 hybridized
carbon of Y; and A is a covalently linked residue of a pharmacologically
active molecule; and x is 1-25, and if POLY is a linear polyethylene
glycol, x=1, and Y is a linear alkyl chain, the POLY has a molecular
weight of at least 500, wherein when S--W is the disulfide linkage
ortho-pyridyl disulfide and POLY is a linear polyethylene glycol, at
least one R1 on a carbon adjacent to said disulfide linkage is lower
alkyl.

2. The conjugate of claim 1, wherein said conjugate is water soluble.

3. The conjugate of claim 2, wherein x is 1.

4. The conjugate of claim 2, wherein x is 2.

5. The conjugate of claim 1, wherein POLY is a polyethylene glycol.

6. The conjugate of claim 5, wherein said polyethylene glycol has a
molecular weight of 148 to about 100,000 Daltons and a morphology
selected from linear, branched, forked, and multiarmed.

7. The conjugate of claim 2, wherein each of R1 and R2 is
independently selected from hydrogen and lower alkyl.

8. The conjugate of claim 9, wherein each of R1 and R2 is
independently selected from hydrogen and methyl.

9. The conjugate of claim 8, wherein each of R1 and R2 is
hydrogen.

10. The conjugate of claim 9, wherein each of R1 and R2 is
hydrogen with the exception of R1 on a carbon adjacent said sulfur
atom, said R1 being lower alkyl.

11. The conjugate of claim 2, wherein two groups R1 and R2 on
different carbon atoms are linked to form a cycloalkyl or aryl group.

12. The conjugate of claim 1, wherein said molecule has a reactive thiol
group in its unconjugated form and is selected from the group consisting
of proteins, peptides, and small molecules.

13. The conjugate of claim 1, comprising the structure:
A-S--S--Y-POLY-Y--S--S-A.

14. The conjugate of claim 13, wherein the Y groups are identical.

15. The conjugate of claim 1, in combination with a pharmaceutical
excipient.

16. The conjugate of claim 15, wherein said excipient is an aqueous
carrier.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent application Ser.
No. 12/943,740, filed Nov. 10, 2010, which is a continuation of U.S.
patent application Ser. No. 11/316,051, filed Dec. 21, 2005, now U.S.
Pat. No. 7,851,565, which claims priority to U.S. Provisional Application
Ser. No. 60/639,823, filed Dec. 21, 2004, and U.S. Provisional
Application Ser. No. 60/705,968, filed Aug. 4, 2005, each of which is
hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

[0002] The present invention relates to stabilized polymeric thiol
reagents derived from water-soluble polymers such as polyethylene glycol.
In particular, the invention relates to the polymeric thiol reagents,
conjugates thereof, and methods for utilizing such conjugates.

BACKGROUND OF THE INVENTION

[0003] Due to recent advances in biotechnology, therapeutic proteins and
other biomolecules, e.g. antibodies and antibody fragments, can now be
prepared on a large scale, making such biomolecules more widely
available. Unfortunately, the clinical usefulness of potential
therapeutic biomolecules in unmodified form is often hampered by their
rapid proteolytic degradation, instability upon manufacture, storage or
administration, or by their immunogenicity. These deficiencies can often
be overcome by covalent attachment of a water-soluble polymer, such as
polyethylene glycol (PEG). See, for example, Abuchowski, A. et al., J.
Biol. Chem. 252(11):3579 (1977); Davis, S. et al., Clin. Exp Immunol.
46:649-652 (1981). The biological properties of PEG-modified proteins,
also referred to as PEG-conjugates or PEGylated proteins, have been
shown, in many cases, to be considerably improved over those of their
non-PEGylated counterparts (Herman et al., Macromol. Chem. Phys.
195:203-209 (1994)). Polyethylene glycol-modified proteins have been
shown to possess longer circulatory times in the body, due to increased
resistance to proteolytic degradation, and also to possess increased
thermostability (Abuchowski, A. et al., J. Biol. Chem. 252:3582-3586
(1977). A similar increase in bioefficacy is observed with other
biomolecules, e.g. antibodies and antibody fragments (Chapman, A., Adv.
Drug Del. Rev. 54:531-545 (2002)).

[0004] Polyethylene glycol having activated end groups suitable for
reaction with amino groups are commonly used for modification of
proteins. Such activated PEGs or "polymeric reagents" include
PEG-aldehydes (Harris, J. M. and Herati, R. S., Polym Prepr. (Am. Chem.
Soc., Div. Polym. Chem.) 32(1):154-155 (1991)), mixed anhydrides,
N-hydroxysuccinimide esters, carbonylimidazolides, and chlorocyanurates
(Herman, S. et al., Macromol. Chem. Phys. 195:203-209 (1994)). In some
cases, however, polymer attachment through protein amino groups can be
undesirable, such as when derivatization of specific lysine residues
inactivates the protein (Suzuki, T. et al., Biochimica et Biophysica Acta
788:248-255 (1984)). Therefore, it would be advantageous to have
additional methods for the modification of a protein by PEG using another
target amino acid for attachment, such as cysteine. Attachment to protein
thiol groups on cysteine offers an advantage in that cysteines are
typically less abundant in proteins than lysines, thus reducing the
likelihood of protein deactivation upon conjugation to these
thiol-containing amino acids. Thiol-selective activated polymers are
described, for example, in commonly owned PCT publication no. WO
2004/063250.

[0005] Polymeric thiol derivatives, and specifically PEG thiols, are one
type of thiol-selective activated polymer. However, many prior art
polymeric thiols suffer from being highly susceptible to oxidative
coupling to form disulfides, a degradative process that reduces the
active component and adds difficult-to-remove impurities. The latter can
be particularly problematic in the preparation of bioconjugates from
these materials. Therefore, it would be advantageous to provide polymeric
thiol reagents having enhanced stability over prior art reagents.

SUMMARY OF THE INVENTION

[0006] In one aspect, the invention provides a water-soluble activated
polymer, also referred to as a "polymeric reagent," having the structure

POLY-[Y--S--W]x

wherein:

[0007] POLY is a water soluble polymer;

[0008] Y is a divalent linking group, containing at least four carbon
atoms, consisting of a saturated or unsaturated hydrocarbon backbone
which is three to eight carbon atoms in length and has substituents which
are independently selected from hydrogen, lower alkyl, lower alkenyl, and
non-interfering substituents as defined herein, where two such alkyl
and/or alkenyl substituents on different carbon atoms of the backbone may
be linked so as to form a cycloalkyl, cycloalkenyl, or aryl group;

[0009] S is a sulfur atom attached to an sp3 hybridized carbon of Y;

[0010] x is 1 to 25; and

[0011] S--W is a thiol, protected thiol, or thiol-reactive thiol
derivative. In one embodiment, S--W is a thiol-reactive derivative, such
as ortho-pyridyl disulfide (OPSS).

[0012] When x is 2, the reagent is a difunctional polymeric reagent, such
as described further below, and it may have a linear or a "forked"
morphology, as described herein. The polymeric reagent may also have a
"multiarmed" morphology, as described herein, particularly when x is 3 or
greater. In selected embodiments, x is 1 to 8, 1 to 6, or 1 to 4; in
further embodiments, x is 1 or 2, or x is 1. The POLY component of the
disclosed polymeric reagents can itself have a morphology selected from
the group consisting of linear, branched, multi-armed, and combinations
thereof, as described further herein.

[0013] In a particular embodiment, when POLY is a linear polyethylene
glycol and Y is a linear alkyl chain, POLY has a molecular weight of at
least 500.

[0014] In further embodiments, POLY has a molecular weight of at least
1000, or at least 2000. As an upper range, POLY has a molecular weight of
not greater than 300,000 Da.

[0015] The "hydrocarbon backbone" of the linking group Y is more
particularly defined as the shortest contiguous carbon chain connecting
POLY and S. When the backbone of Y is unsaturated, it is preferably
monounsaturated, i.e. having a single double or triple carbon-carbon
bond. Preferably, the spacer group Y, including backbone and
substituents, is monounsaturated or, more preferably, fully saturated. In
another embodiment, the spacer group Y, including backbone and
substituents, consists of saturated and aromatic portions.

[0016] Preferably, the backbone is saturated. For example, Y may be of the
form --(CR1R2)n--, where n is 3 to 8, and each of R1
and R2 is independently selected from hydrogen, lower alkyl, lower
alkenyl, and a non-interfering substituent, where two groups R1 and
R2 on different carbon atoms may be linked to form a cycloalkyl or
aryl group. In selected embodiments, n is 3 to 6, n is 4 to 6, or n=4,
and each of R1 and R2 is independently selected from hydrogen
and lower alkyl, where lower alkyl is preferably methyl or ethyl.

[0017] In further selected embodiments, Y is selected from the group
consisting of C4-C8 alkylene, C5-C8 cycloalkylene,
and combinations thereof, any of which may include one or more
non-interfering substituents.

[0018] Preferably, at most one or two non-interfering substituents,
selected from the group consisting of C3-C6 cycloalkyl, halo,
cyano, lower alkoxy, and phenyl, and preferably selected from methoxy,
ethoxy, fluoro, and chloro, are included. In one embodiment, no
heteroatom-containing substituents are present; that is, the linking Y
consists of carbon and hydrogen.

[0019] In one preferred embodiment, each of R1 and R2 is
hydrogen with respect to the n iterations of --(CR1R2)--; in
another preferred embodiment, each of R1 and R2 is hydrogen
with the exception of R1 on a carbon adjacent said sulfur atom, said
R1 being lower alkyl, preferably methyl or ethyl
(α-branching). In one embodiment, the α-branch group is
methyl.

[0020] In embodiments of Y where Y is --(CR1R2)n-- and two
groups R1 and R2 on different carbon atoms are linked to form a
cycloalkyl, cycloalkenyl, or aryl group, the cycloalkyl group is
preferably a cyclopentyl or cyclohexyl group. Preferably, S is linked to
an sp3 hybridized acyclic carbon of Y in such embodiments.

[0021] As noted above, POLY may be a polyethylene glycol (PEG). Such a PEG
typically has a molecular weight of 148 (e.g. a trimer plus linking
oxygen atom) to about 200 to about 100,000 Daltons. In selected
embodiments, the polyethylene glycol has a molecular weight from about
200 to about 40,000 Daltons. Representative molecular weights include,
for example, 500, 1000, 2000, 2500, 3500, 5000, 7500, 10000, 15000,
20000, 25000, 30000, and 40000 Daltons. The PEG component of the
disclosed reagents can itself have a morphology selected from the group
consisting of linear, branched, multi-armed, and combinations thereof, as
described further herein.

[0022] Accordingly, the invention provides a water soluble polymeric
reagent comprising the structure:

PEG-[Y--S--W]x

wherein:

[0023] PEG is a polyethylene glycol polymer;

[0024] Y is a divalent linking group consisting of a saturated or
unsaturated hydrocarbon backbone which is three to eight carbon atoms in
length and has substituents which are independently selected from
hydrogen, lower alkyl, lower alkenyl, and non-interfering substituents as
defined herein, where two such alkyl and/or alkenyl substituents on
different carbon atoms of the backbone may be linked so as to form a
cycloalkyl, cycloalkenyl, or aryl group;

[0025] S is a sulfur atom attached to an sp3 hybridized carbon of Y;

[0026] x is 1 to 25;

[0027] S--W is a thiol, protected thiol, or thiol-reactive thiol
derivative; and

[0028] PEG has a molecular weight of at least 500 when PEG is linear, x is
1, and Y is a linear alkyl chain.

[0029] As stated above, when x is 2, the reagent is a difunctional
polymeric reagent, such as described further below, and it may have a
linear or a "forked" morphology, as described herein. The polymeric
reagent may also have a "multiarmed" morphology, as described herein,
particularly when x is 3 or greater. In selected embodiments, x is 1 to
8, 1 to 6, or 1 to 4; in further embodiments, x is 1 or 2, or x is 1. The
PEG component of the disclosed reagents can itself have a morphology
selected from the group consisting of linear, branched, multi-armed, and
combinations thereof, as described further herein.

[0030] In preferred embodiments, PEG has a molecular weight of at least
148, at least 200, at least 500, at least 1000, or at least 2000, up to
about 100,000 Daltons, including the various ranges noted above, and a
morphology selected from linear, branched, forked, and multiarmed. S--W
is preferably a thiol-reactive thiol derivative, and more preferably
ortho-pyridyl disulfide (OPSS).

[0031] The "hydrocarbon backbone" of the linking group Y is more
particularly defined as the shortest contiguous carbon chain connecting
PEG and S. Preferably, the backbone of the linking group Y is saturated,
such that Y has the formula --(CR1R2)n-, where n is 3 to 8, and
each of R1 and R2 is independently selected from hydrogen,
lower alkyl, lower alkenyl, and a non-interfering substituent, where two
groups R1 and R2 on different carbon atoms may be linked to
form a cycloalkyl or aryl group. In selected embodiments, n is 3 to 6, n
is 4 to 6, or n=4, and each of R1 and R2 is independently
selected from hydrogen and methyl.

[0032] In further selected embodiments, Y is selected from the group
consisting of C3-C8 alkylene, C5-C8 cycloalkylene,
and combinations thereof, any of which may include one or more
non-interfering substituents, as described above. Preferably, at most one
or two non-interfering substituents, selected from the group consisting
of C3-C6 cycloalkyl, halo, cyano, lower alkoxy, and phenyl, and
preferably selected from methoxy, ethoxy, fluoro, and chloro, are
included. In one embodiment, no heteroatom-containing substituents are
present; that is, the linking group Y consists of carbon and hydrogen.

[0033] As above, in one preferred embodiment, each of R1 and R2
is hydrogen with respect to the n iterations of --(CR1R2)--; in
another preferred embodiment, each of R1 and R2 is hydrogen
with the exception of R1 on a carbon adjacent said sulfur atom, said
R1 being lower alkyl, preferably methyl or ethyl
(α-branching). In one embodiment, the α-branch group is
methyl.

[0034] In embodiments of Y where Y is --(CR1R2)n- and two groups
R1 and R2 on different carbon atoms are linked to form a
cycloalkyl, cycloalkenyl, or aryl group, the cycloalkyl group is
preferably a cyclopentyl or cyclohexyl group. Preferably, S is linked to
an sp3 hybridized acyclic carbon of Y in such embodiments.

[0035] In an exemplary reagent of the form PEG-Y--S--W, Y is
--(CR1R2)n- where each of R1 and R2 is hydrogen and n
is 4, S--W is ortho-pyridyl disulfide (OPSS), and PEG is a
methoxy-terminated polyethylene glycol (mPEG). The mPEG preferably has a
molecular weight in the range of 5000 to 30000 Da. In further exemplary
reagents, PEG and SW are similarly defined, n is 3 or 4, and each of
R1 and R2 is hydrogen with the exception of R1 on a carbon
adjacent said sulfur atom, said R1 being methyl (i.e., Y is
--CH2CH2CH(CH3)-- or
--CH2CH2CH2CH(CH3)--).).

[0036] The water soluble polymeric reagents may have a polyfunctional
structure, as shown:

POLY-[Y--S--W]x

wherein:

[0037] POLY is a water soluble polymer;

[0038] x is 2 to 25;

[0039] each Y is a divalent linking group, having at least four carbon
atoms, consisting of a saturated or unsaturated hydrocarbon backbone
which is three to ten, preferably three to eight, carbon atoms in length
and has substituents which are independently selected from hydrogen,
lower alkyl, lower alkenyl, and non-interfering substituents as defined
herein, where two such alkyl and/or alkenyl substituents on different
carbon atoms of the backbone may be linked so as to form a cycloalkyl,
cycloalkenyl, or aryl group;

[0040] each S is a sulfur atom attached to an sp3 hybridized carbon
of the adjacent Y; and

[0041] each S--W is a independently a thiol, protected thiol, or
thiol-reactive thiol derivative.

[0042] Preferably, the two or more Y groups are identical; the two or more
W groups are also typically identical. Alternatively, particularly in a
difunctional reagent (x=2), the two SW's may be different; e.g. one SW is
a thiol or protected thiol while the other is a thiol-reactive
derivative, or one SW is a thiol or thiol-reactive derivative while the
other is a protected thiol.

[0043] As noted above, when x is 2, the polymeric reagent may have a
linear or a "forked" morphology, as described herein. The polymeric
reagent may also have a "multiarmed" morphology, as described herein,
particularly when x is 3 or greater. In selected embodiments, x is 2 to
8, 2 to 6, or 2 to 4; in one embodiment, x is 2. The POLY component of
the disclosed reagents can itself have a morphology selected from the
group consisting of linear, branched, multi-armed, and combinations
thereof, as described further herein.

[0044] As above, the backbone of Y is preferably saturated, such that each
Y is a linker having at least four carbon atoms and having the formula
--(CR1R2)n-, where n is 3 to 10, preferably 3 to 8, and each of
R1 and R2 is independently selected from hydrogen, lower alkyl,
lower alkenyl, and a non-interfering substituent, where two groups
R1 and R2 on different carbon atoms may be linked to form a
cycloalkyl or aryl group. Further preferred embodiments of Y, and of
POLY, are generally as defined above for the monomeric reagent
POLY-Y--S--W.

[0045] The corresponding PEG-based polyfunctional polymeric reagents have
the structure:

PEG-[Y--S--W]x

wherein:

[0046] PEG is polyethylene glycol polymer;

[0047] x is 2 to 25;

[0048] each Y is a divalent linking group consisting of a saturated or
unsaturated hydrocarbon backbone which is three to ten, preferably three
to eight, carbon atoms in length and has substituents which are
independently selected from hydrogen, lower alkyl, lower alkenyl, and
non-interfering substituents as defined herein, where two such alkyl
and/or alkenyl substituents on different carbon atoms of the backbone may
be linked so as to form a cycloalkyl, cycloalkenyl, or aryl group;

[0049] each S is a sulfur atom attached to an sp3 hybridized carbon
of the adjacent Y; and

[0050] each S--W is a independently a thiol, protected thiol, or
thiol-reactive thiol derivative.

[0051] Preferably, the multiple Y groups are identical; the multiple W
groups are also typically identical. The backbone of Y is preferably
saturated, such that each Y is a linker having the formula
--(CR1R2)n-, where n is 3 to 10, preferably 3 to 8, and each of
R1 and R2 is independently selected from hydrogen, lower alkyl,
lower alkenyl, and a non-interfering substituent, where two groups
R1 and R2 on different carbon atoms may be linked to form a
cycloalkyl or aryl group.

[0052] Further preferred embodiments of Y, and of PEG, are generally as
defined above for PEG-[Y--S--W]x. In an exemplary difunctional
reagent of the form W--S--Y-PEG-Y--S--W, each Y is
--(CR1R2)n-- where each of R1 and R2 is hydrogen
and n is 4, S--W is ortho-pyridyl disulfide (OPSS), and each PEG is a
methoxy-terminated polyethylene glycol (mPEG). The mPEG preferably has a
molecular weight in the range of 1000 to 5000 Da, e.g. about 2000 or
about 3400 Da. In a further exemplary reagent, PEG and SW are similarly
defined, n is 3 or 4, and each of R1 and R2 is hydrogen with
the exception of R1 on a carbon adjacent said sulfur atom, said
R1 being methyl.

[0053] In a related aspect, the invention provides a polymer conjugate
comprising the structure:

POLY-[Y--S--S-A]x

wherein:

[0054] POLY is a water soluble polymer;

[0055] x is 1 to 25;

[0056] Y is a divalent linking group consisting of a saturated or
unsaturated hydrocarbon backbone which is three to ten, preferably three
to eight, carbon atoms in length and has substituents which are
independently selected from hydrogen, lower alkyl, lower alkenyl, and
non-interfering substituents as defined herein, where two such alkyl
and/or alkenyl substituents on different carbon atoms of the backbone may
be linked so as to form a cycloalkyl, cycloalkenyl, or aryl group;

[0057] S--S is a disulfide group attached to an sp3 hybridized carbon
of Y; and

[0058] A is a covalently linked residue (as defined herein) of a
pharmacologically active molecule.

[0059] In selected embodiments, x is 1 to 8, 1 to 6, or 1 to 4; in further
embodiments, x is 1 or 2, or x is 1. When x is 2, the conjugate may have
a linear or a "forked" morphology, as described herein. The conjugate may
also have a "multiarmed" morphology, as described herein, particularly
when x is 3 or greater. The POLY component of the conjugate can itself
have a morphology selected from the group consisting of linear, branched,
multi-armed, and combinations thereof, as described further herein.

[0060] Preferably, the multiple Y groups are identical. The hydrocarbon
backbone of Y is preferably saturated, with Y having the formula
--(CR1R2)n-, where n is 3 to 10, preferably 3 to 8, each of
R1 and R2 is independently selected from hydrogen, alkyl,
alkenyl, and a non-interfering substituent, and where two groups R1
and R2 on different carbon atoms may be linked to form a cycloalkyl
or aryl group. More preferably, Y is selected from the group consisting
of C3-C8 alkylene, C5-C8 cycloalkylene, aryl, and
combinations thereof, any of which may include one or more
non-interfering substituents. In one embodiment, Y has at least four
carbon atoms.

[0061] In further embodiments, Y is a linear or branched alkylene having
the formula --(CR1R2)n-, where n is 3 to 10, and each of
R1 and R2 is independently selected from hydrogen, lower alkyl,
lower alkenyl, and a non-interfering substituent. More preferably, n is 3
to 8, or 3 to 6, and each of R1 and R2 is independently
selected from hydrogen and methyl. In selected embodiments, each of
R1 and R2 is hydrogen. In another preferred embodiment, each of
R1 and R2 is hydrogen with the exception of R1 on a carbon
adjacent said disulfide linkage, said R1 being lower alkyl, e.g.
methyl or ethyl.

[0062] Other embodiments include those in which Y is
--(CR1R2)n-, where n is 3 to 10, preferably 3 to 8, and two
groups R1 and R2 on different carbon atoms are linked to form a
cycloalkyl or aryl group, preferably a cycloalkyl such as cyclopentyl or
cyclohexyl group.

[0063] The water soluble polymer POLY preferably has a molecular weight of
at least 500, or at least 1000. The molecular weight of POLY is typically
greater than 200 and less than about 300K Daltons, preferably less than
about 200K Daltons, and more preferably less than about 100K Daltons. In
one embodiment, POLY is a polyethylene glycol, preferably having a
molecular weight of 148 to about 200 to about 100,000 Daltons, and a
morphology selected from linear, branched, forked, and multiarmed. In
selected embodiments, the polyethylene glycol has a molecular weight from
about 200 to about 40,000 Daltons. Representative molecular weights
include, for example, 500, 1000, 2500, 3500, 5000, 7500, 10000, 15000,
20000, 25000, 30000, and 40000 Daltons.

[0064] The molecule conjugated to the water soluble polymer, represented
by A in its conjugated form, has a reactive thiol group in its
unconjugated form and is preferably selected from the group consisting of
proteins, peptides, and small molecules, typically small organic
molecules.

[0065] The conjugate is preferably itself water soluble. The conjugate may
be provided in or with a suitable pharmaceutical excipient, such as an
aqueous carrier, for therapeutic use.

[0066] In a related aspect, the invention provides a method for delivering
a pharmacologically active molecule having a reactive thiol group to a
subject, by administering to the subject a conjugate as described above,
in a pharmaceutically acceptable carrier. The conjugate is typically
prepared by conjugating the molecule with any of the water soluble
polymeric reagents described herein.

[0067] The hydrocarbon-based segment(s), Y, in the activated polymeric
reagents of the invention, being hydrophobic in nature, are effective to
reduce the tendency towards dimerization of these reagents, relative to
prior art reagents in which the thiol is linked to a heteroatom in the
polymer segment (or in a linking moiety) by, for example, a two-carbon
linkage. Branching of Y at the carbon adjacent to the sulfur atom
(α-branching) is further effective to reduce dimerization. The
hydrocarbon-based segment Y also reduces cleavage, e.g. enzymatic
cleavage in vivo, of the adjacent disulfide bond, in conjugates formed
using these reagents.

[0068] The reagents disclosed herein are further characterized as being
"linkerless" reagents; that is, the water-soluble polymer, preferably a
PEG, is directly linked to the hydrocarbon-based spacer group Y. The
absence of heteroatom-containing linkages, such as amides, esters, or
carbamates, between the active conjugating functionality, i.e. the thiol
or protected thiol, and the polymer reduces the potential for degradation
of the resulting conjugate. Moreover, the presence of these linkages,
such as amides, in such reagents can trigger a deleterious immune
response. This potential is eliminated or greatly reduced by use of the
current "linkerless" reagents.

[0069] As shown in Example 2 herein, a polymeric thiol reagent of the
invention was more stable under synthetic conditions than a corresponding
reagent having only a two-carbon linker between the hydrophilic polymer
(PEG) and thiol group. This increased stability is also exhibited in
Example 9 and comparative Example 10. The conjugation behavior of the
polymeric thiol reagent of the invention was similar to that observed for
a polymeric reagent (a maleimide-terminated polymer) known not to
dimerize, as also shown in Example 2.

[0070] These and other objects and features of the invention will become
more fully apparent when the following detailed description of the
invention is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0071] FIG. 1 shows a PAGE analysis of a conjugation reaction between a
polymeric reagent of the invention, designated mPEG5000-4C-OPSS, as
described in Examples 1-2, with reduced BSA, and the corresponding
conjugation reaction of the polymeric reagent mPEG5000-maleimide
with reduced BSA: Lane 1, standards; Lane 2, reduced BSA; Lane 3,
conjugation with mPEG-MAL; Lane 4, conjugation with mPEG-4C-OPSS. The gel
is stained with SimplyBlue Safe Stain; and

[0073] The following terms as used herein have the meanings indicated. As
used in the specification, and in the appended claims, the singular forms
"a," "an," "the," include plural referents unless the context clearly
dictates otherwise.

[0074] "PEG" or "polyethylene glycol," as used herein, is meant to
encompass any water soluble poly(ethylene oxide). Typically, PEGs for use
in the present invention will comprise one of the two following
structures:
--CH2CH2--O--(CH2CH2O)m--CH2CH2-- or
--O(CH2CH2O)m--, where m is generally from 2 to about
6000, more typically 4 or 5 to about 2500. In a broader sense, "PEG" can
refer to a polymer that contains a majority, i.e., greater than 50%, of
subunits that are --CH2CH2O--. Preferably, greater than 75%,
greater than 95%, or substantially all of the monomeric subunits are
--CH2CH2O--. The terminal groups and architecture of the
overall PEG may vary. One terminus of the PEG may contain an end-capping
group, which is generally a carbon-containing group comprised of 1-20
carbons and is preferably selected from alkyl, alkenyl, alkynyl, aryl,
aralkyl, cycloalkyl, heterocyclo, and substituted forms of any of the
foregoing. The end-capping group can also be a silane. An end-capping
group is one that does not readily undergo chemical transformation under
typical synthetic reaction conditions. Most preferred are alkyl (alkoxy)
or aralkyl (aralkoxy) capping groups, such as methyl, ethyl or benzyl.

[0075] The end-capping group can also advantageously comprise a
phospholipid. When the polymer has an end-capping group such as a
phospholipid, unique properties (such as the ability to form organized
structures with similarly end-capped polymers) are imparted to the
polymer. Exemplary phospholipids include, without limitation, those
selected from the class of phospholipids called phosphatidylcholines.
Specific phospholipids include, without limitation, those selected from
the group consisting of dilauroyl phosphatidylcholine, dioleyl
phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl
phosphatidylcholine, behenoyl phosphatidylcholine, arachidoyl
phosphatidylcholine, and lecithin.

[0076] The end-capping group can also advantageously comprise a detectable
label. Such labels include, without limitation, fluorescers,
chemiluminescers, moieties used in enzyme labeling, colorimetric (e.g.,
dyes), metal ions, radioactive moieties, and the like.

[0077] The PEG may also be terminated with a functional group, such as
those described below, preferably in protected form.

[0078] Specific PEG forms for use in the invention include PEGs having a
variety of molecular weights, structures or geometries (e.g., branched,
linear, forked, multiarmed).

[0079] "Branched," in reference to the geometry or overall structure of a
polymer, refers to polymer having 2 or more polymer "arms." A branched
polymer may possess 2 polymer arms, 3 polymer arms, 4 polymer arms, 6
polymer arms, 8 polymer arms or more. One particular type of highly
branched polymer is a dendritic polymer or dendrimer, that for the
purposes of the invention, is considered to possess a structure distinct
from that of a branched polymer. A "branch point" refers to a bifurcation
point comprising one or more atoms at which a polymer splits or branches
from a linear structure into one or more additional polymer arms.

[0080] A "dendrimer" is a globular, size monodisperse polymer in which all
bonds emerge radially from a central focal point or core with a regular
branching pattern and with repeat units that each contribute a branch
point. Dendrimers exhibit certain dendritic state properties such as core
encapsulation, making them unique from other types of polymers.

[0081] "Water-soluble," in the context of a polymer of the invention or a
"water-soluble polymer segment", is any segment or polymer that is
soluble in water at room temperature. Typically, a water-soluble polymer
or segment will transmit at least about 75%, more preferably at least
about 95% of light, transmitted by the same solution after filtering. On
a weight basis, a water-soluble polymer or segment thereof will
preferably be at least about 35% (by weight) soluble in water, more
preferably at least about 50% (by weight) soluble in water, still more
preferably about 70% (by weight) soluble in water, and still more
preferably about 85% (by weight) soluble in water. It is most preferred,
however, that the water-soluble polymer or segment is about 95% (by
weight) soluble in water or completely soluble in water.

[0082] "Molecular mass" or "molecular weight" of a polymer, unless
otherwise specified, refers to number average molecular weight. Number
average molecular weight (Mn) is defined as
ΣNiMi/ΣNi wherein Ni is the number of
polymer molecules (or the number of moles of those molecules) having
molecular weight Mi. The number average molecular weight of a
polymer can be determined by osmometry, end-group titration, and
colligative properties. Weight average molecular weight is defined as
ΣNiMi2/ΣNiMi, where Ni is again
the number of molecules of molecular weight Mi. The weight average
molecular weight can be determined by light scattering, small angle
neutron scattering (SANS), X-ray scattering, and sedimentation velocity.

[0083] The polymers of the invention, or employed in the invention, may be
polydisperse; i.e., the number average molecular weight and weight
average molecular weight of the polymers are not equal. However, the
polydispersity values, expressed as a ratio of weight average molecular
weight (Mw) to number average molecular weight (Mn),
(Mw/Mn), are generally low; that is, less than about 1.2,
preferably less than about 1.15, more preferably less than about 1.10,
still more preferably less than about 1.05, yet still most preferably
less than about 1.03, and most preferably less than about 1.025.

[0084] The term "reactive" or "activated" refers to a functional group
that reacts readily or at a practical rate under conventional conditions
of organic synthesis. This is in contrast to those groups that either do
not react or require strong catalysts or impractical reaction conditions
in order to react (i.e., a "nonreactive" or "inert" group).

[0085] "Not readily reactive" or "inert," with reference to a functional
group present on a molecule in a reaction mixture, indicates that the
group remains largely intact under conditions effective to produce a
desired reaction in the reaction mixture.

[0086] A "protecting group" is a moiety that prevents or blocks reaction
of a particular chemically reactive functional group in a molecule under
certain reaction conditions. The term may also refer to the protected
form of a functional group. The protecting group will vary depending upon
the type of chemically reactive group being protected as well as the
reaction conditions to be employed and the presence of additional
reactive or protecting groups in the molecule. Functional groups which
may be protected include, by way of example, carboxylic acid groups,
amino groups, hydroxyl groups, thiol groups, carbonyl groups and the
like. Representative protected forms of such functional groups include,
for carboxylic acids, esters (such as a p-methoxybenzyl ester), amides
and hydrazides; for amino groups, carbamates (such as tert-butoxycarbonyl
or fluorenylmethoxycarbonyl) and amides; for hydroxyl groups, ethers and
esters; for thiol groups, thioethers and thioesters; for carbonyl groups,
acetals and ketals; and the like. Such protecting groups are well-known
to those skilled in the art and are described, for example, in T. W.
Greene and G. M. Wuts, Protecting Groups in Organic Synthesis, Third
Edition, Wiley, New York, 1999, and references cited therein.

[0087] A "thiol-reactive derivative" of a thiol refers to a thiol
derivative which can react with another thiol, preferably under
conditions of moderate temperature and neutral or physiological pH, to
form a disulfide linkage. Preferably, the reaction forms only stable
byproducts. Typical examples of such derivatives are ortho-pyridyl
disulfides and TNB-thiol derivatives (where TNB is 5-thio-2-nitrobenzoic
acid). See e.g. Hermanson, Bioconjugate Techniques, Academic Press, 1996,
pp 150-152.

[0088] As used herein, the term "functional group" or any synonym thereof
is meant to encompass protected or derivatized forms thereof. Similarly,
the term "thiol reagent" or "polymeric thiol" encompasses protected or
derivatized thiol reagents or polymeric protected or derivatized thiols
(such as polymer-OPSS).

[0089] A "physiologically cleavable" or "hydrolysable" or "degradable"
bond is a relatively weak bond that reacts with water (i.e., is
hydrolyzed) under physiological conditions. The tendency of a bond to
hydrolyze in water will depend not only on the general type of linkage
connecting two central atoms but also on the substituents attached to
these central atoms. Appropriate hydrolytically unstable or weak linkages
include but are not limited to carboxylate ester, phosphate ester,
anhydrides, acetals, ketals, acyloxyalkyl ether, imines, orthoesters,
peptides and oligonucleotides, thioesters, thiolesters, and carbonates.
An "enzymatically degradable linkage" means a linkage that is subject to
degradation by one or more enzymes.

[0090] "Substantially" or "essentially" means nearly totally or
completely, for instance, 95%, 99% or greater of some given quantity.

[0091] "Alkyl" refers to a hydrocarbon chain, typically ranging from about
1 to 20 atoms in length. Such hydrocarbon chains are preferably but not
necessarily saturated and may be branched or, preferably, linear
(unbranched). Exemplary alkyl groups include ethyl, propyl, butyl,
pentyl, 2-methylbutyl, 2-methylpropyl (isobutyl), 3-methylpentyl, and the
like. As used herein, "alkyl" includes cycloalkyl when three or more
carbon atoms are referenced. "Alkylene" refers to a divalent alkyl group,
e.g.--(CH2)x--.

[0092] "Lower alkyl" refers to an alkyl group containing from 1 to 6
carbon atoms, more preferably 1 to 4 carbon atoms, as exemplified by
methyl, ethyl, n-butyl, isopropyl, and t-butyl.

[0093] "Cycloalkyl" refers to a saturated or unsaturated cyclic
hydrocarbon chain, including bridged, fused, or Spiro cyclic compounds,
preferably made up of 3 to about 12 carbon atoms, more preferably 3 to
about 8. "Cycloalkylene" refers to a divalent cycloalkyl group.

[0094] As used herein, "alkenyl" refers to a branched or unbranched
hydrocarbon group having 2 to 15 carbon atoms and containing at least one
double bond, such as ethenyl, n-propenyl, isopropenyl, n-butenyl,
isobutenyl, octenyl, decenyl, tetradecenyl, and the like.

[0095] The term "alkynyl" as used herein refers to a branched or
unbranched hydrocarbon group having 2 to 15 atoms and containing at least
one triple bond, such as ethynyl, n-propynyl, isopentynyl, n-butynyl,
octynyl, decynyl, and so forth.

[0097] "Aryl" refers to a substituted or unsubstituted monovalent aromatic
radical having a single ring (e.g., phenyl) or two condensed or fused
rings (e.g., naphthyl). Multiple aryl rings may also be unfused (e.g.
biphenyl). The term includes heteroaryl groups, which are aromatic ring
groups having one or more nitrogen, oxygen, or sulfur atoms in the ring,
such as furyl, pyrrole, pyridyl, and indole.

[0098] "Aralkyl" refers to an alkyl, preferably lower (C1-C4,
more preferably C1-C2) alkyl, substituent which is further
substituted with an aryl group; examples are benzyl and phenethyl.
"Aralkoxy" refers to a group of the form --OR where R is aralkyl; one
example is benzyloxy.

[0099] A "heterocycle" refers to a ring, preferably a 5- to 7-membered
ring, whose ring atoms are selected from the group consisting of carbon,
nitrogen, oxygen and sulfur. Preferably, the ring atoms include 3 to 6
carbon atoms. Examples of aromatic heterocycles (heteroaryl) are given
above; non-aromatic heterocycles include, for example, pyrrolidine,
piperidine, piperazine, and morpholine.

[0100] A "substituted" group or moiety is one in which a hydrogen atom has
been replaced with a non-hydrogen atom or group, which is preferably a
non-interfering substituent.

[0101] "Non-interfering substituents" are those groups that, when present
in a molecule, are typically non-reactive with other functional groups
contained within the molecule.

[0103] By "residue" is meant the portion of a molecule remaining after
reaction with one or more molecules. For example, a biologically active
molecule residue in a polymer conjugate of the invention typically
corresponds to the portion of the biologically active molecule up to but
excluding the covalent linkage resulting from reaction of a reactive
group on the biologically active molecule with a reactive group on a
polymer reagent.

[0104] The term "conjugate" refers to an entity formed as a result of
covalent attachment of a molecule, e.g., a biologically active molecule,
to a reactive polymer molecule, preferably a poly(ethylene glycol).

[0105] Each of the terms "drug," "biologically active molecule,"
"biologically active moiety," "biologically active agent",
"pharmacologically active agent", and "pharmacologically active
molecule", when used herein, means any substance which can affect any
physical or biochemical property of a biological organism, where the
organism may be selected from viruses, bacteria, fungi, plants, animals,
and humans. In particular, as used herein, biologically active molecules
include any substance intended for diagnosis, cure mitigation, treatment,
or prevention of disease in humans or other animals, or to otherwise
enhance physical or mental well-being of humans or animals. Examples of
biologically active molecules include, but are not limited to, peptides,
proteins, enzymes, small molecule drugs, dyes, lipids, nucleosides,
oligonucleotides, polynucleotides, nucleic acids, cells, viruses,
liposomes, microparticles and micelles. Classes of biologically active
agents that are suitable for use with the invention include, but are not
limited to, antibiotics, fungicides, anti-viral agents, anti-inflammatory
agents, anti-tumor agents, cardiovascular agents, anti-anxiety agents,
hormones, growth factors, steroidal agents, and the like. Also included
are foods, food supplements, nutrients, nutraceuticals, drugs, vaccines,
antibodies, vitamins, and other beneficial agents.

[0106] "Pharmaceutically acceptable excipient" or "pharmaceutically
acceptable carrier" refers to an excipient that can be included in the
compositions of the invention and that causes no significant adverse
toxicological effects to a patient.

[0107] "Pharmacologically effective amount," "physiologically effective
amount," and "therapeutically effective amount" are used herein to refer
to mean the amount of a polymer-active agent conjugate present in a
pharmaceutical preparation that is needed to provide a desired level of
active agent and/or conjugate in the bloodstream or in the target tissue.
The precise amount will depend upon numerous factors, e.g., the
particular active agent, the components and physical characteristics of
pharmaceutical preparation, intended patient population, patient
considerations, and the like, and can readily be determined by one
skilled in the art, based upon the information provided herein and
available in the relevant literature.

[0108] The term "patient" refers to a living organism suffering from or
prone to a condition that can be prevented or treated by administration
of a biologically active agent or conjugate thereof, and includes both
humans and animals.

[0109] Polymeric Thiol Reagents

[0110] The water-soluble, polymeric reagents of the invention comprise the
structure

POLY-[Y--S--W]x

wherein:

[0111] POLY is a water-soluble polymer segment;

[0112] x is 1 to 25;

[0113] Y is a divalent linking group comprising at least four carbon
atoms, and consisting of a saturated or unsaturated hydrocarbon backbone
which is three to eight carbon atoms in length and has substituents which
are independently selected from hydrogen, lower alkyl, lower alkenyl, and
non-interfering substituents as defined herein, where two such alkyl
and/or alkenyl substituents on different carbon atoms of the backbone may
be linked so as to form a cycloalkyl, cycloalkenyl, or aryl group;

[0114] S is a sulfur atom attached to an sp3 hybridized carbon of Y;

[0115] and S--W is a thiol (i.e. W is H), protected thiol, or
thiol-reactive derivative.

[0116] In one embodiment, S--W is a thiol-reactive derivative, such as
ortho-pyridyl disulfide (OPSS). Protected thiols include, for example,
thioethers, such as S-benzyl or S-trityl ethers, and thioesters.

[0117] The sulfur atom S is attached to an sp3 hybridized carbon atom
of Y, as noted, rather than to an aryl ring or double bond. In one
embodiment, the carbon atom to which the sulfur atom is attached has a
lower alkyl substituent, such as methyl or ethyl (α-branching).

[0118] The "backbone" of the spacer group Y is more particularly defined
as the shortest contiguous carbon chain connecting POLY and S. In one
embodiment, the backbone of Y is saturated. For example, Y may be of the
form --(CR1R2)n--, where n is 3 to 8, preferably 3 to 6,
each of R1 and R2 is independently selected from hydrogen,
lower alkyl, lower alkenyl, and a non-interfering substituent, and where
two groups R1 and R2 on different carbon atoms of
--(CR1R2)n-- may be linked to form a cycloalkyl,
cycloalkenyl, or aryl group. When Y contains a cycloalkyl group, it is
preferably a five- or six-membered cycloalkyl group.

[0119] In selected embodiments, Y is selected from the group consisting of
C3-C8 alkylene and combinations of C3-C8 alkylene
with C5-C8 cycloalkylene or aryl, any of which may include one
or more non-interfering substituents. Preferably, at most one or two
non-interfering substituents, selected from the group consisting of
C3-C6 cycloalkyl, halo, cyano, lower alkoxy, and phenyl, and
preferably selected from methoxy, ethoxy, fluoro, and chloro, are
included. In one embodiment, no heteroatom-containing substituents are
present; that is, Y consists of carbon and hydrogen.

[0120] When the backbone of Y is unsaturated, it is preferably
monounsaturated, i.e. having a single double or triple carbon-carbon
bond. Preferably, the spacer group Y, including backbone and
substituents, is monounsaturated or, more preferably, fully saturated. In
this embodiment, Y may be a fully saturated hydrocarbon.

[0121] In another embodiment, the spacer group Y, including backbone and
substituents, consists of saturated and aromatic portions, preferably
saturated and aromatic hydrocarbon portions.

[0122] In the polymeric reagents, when Y is --(CR1R2)n--,
the polymer segment POLY preferably has a molecular weight of at least
500 Da when each of R1 and R2 is hydrogen with respect to the n
iterations of --(CR1R2)--, particularly when POLY is a linear
PEG and x=1 in the formula above. POLY may further have a molecular
weight greater than 500 Da, greater than 750 Da, or greater than 1000 Da.
A variety of greater molecular weight ranges, up to about 300,000 Da,
more typically up to about 100,000 Da, can be used, as described above.

[0123] When x is 2, the reagent is a difunctional polymeric reagent, such
as described further below, and it may have a linear or a "forked"
morphology, as described herein. The polymeric reagent may also have a
"multiarmed" morphology, as described herein, particularly when x is 3 or
greater. In selected embodiments, x is 1 to 8, 1 to 6, or 1 to 4; in
further embodiments, x is 1 or 2, or x is 1. The POLY component of the
disclosed reagents can itself have a morphology selected from the group
consisting of linear, branched, multi-armed, and combinations thereof, as
described further herein.

[0124] In a preferred embodiment, the water soluble polymer segment is a
polyethylene glycol, such that the reagent has the formula

PEG-[Y--S--W]x

wherein:

[0125] PEG is a poly(ethylene glycol);

[0126] Y is a divalent linking group consisting of a saturated or
unsaturated hydrocarbon backbone which is three to eight carbon atoms in
length and has substituents which are independently selected from
hydrogen, lower alkyl, lower alkenyl, and non-interfering substituents as
defined herein, where two such alkyl and/or alkenyl substituents on
different carbon atoms of the backbone may be linked so as to form a
cycloalkyl, cycloalkenyl, or aryl group;

[0130] The inventors have found that, by including a hydrophobic spacer
group between the water-soluble polymer segment and the thiol group in a
water-soluble polymeric thiol, the tendency of such a molecule to
dimerize to form disulfides is reduced. Yields are accordingly increased
in preparation of such reagents and in their conjugation with other
molecules, as demonstrated below.

[0131] The spacer groups described herein are hydrocarbon-based groups
more three carbons or more in length, which preferably contain at least
four carbon atoms, which may include branching carbons (e.g. an
isobutylene, or 1-methylpropylene, linkage). Although described as
"hydrocarbon based", the spacer group may include a limited number of
non-interfering substituents as defined herein. Preferably, however, the
spacer group consists of carbon and hydrogen.

[0132] In preferred embodiments of the polymeric reagent, Y is a linear or
branched alkylene having the formula --(CR1R2)n--, where n
is 3 to 8, and each of R1 and R2 is independently selected from
hydrogen, lower alkyl, lower alkenyl, and a non-interfering substituent.
Preferably, zero to two, more preferably zero or one, such
non-interfering substituents are included.

[0133] Preferably, n is 4 to 8, more preferably 4 to 6. In one embodiment,
each of R1 and R2 is independently selected from hydrogen and
methyl. In a preferred embodiment, each of R1 and R2 is
hydrogen with respect to the n iterations of --(CR1R2)--; in
another preferred embodiment, each of R1 and R2 is hydrogen
with the exception of R1 on a carbon adjacent said sulfur atom
(α-carbon), said R1 being lower alkyl, preferably methyl or
ethyl (α-branching). In one embodiment, the α-branch group is
methyl.

[0134] In embodiments of Y where Y is --(CR1R2)n--, n is 4
to 8, and two groups R1 and R2 on different carbon atoms are
linked to form a cycloalkyl, cycloalkenyl, or aryl group, the cycloalkyl
group is preferably a cyclopentyl or cyclohexyl group. In such
embodiments, the sulfur atom is preferably attached to a non-cyclic
carbon of Y.

[0135] Exemplary spacer groups Y having a saturated backbone include the
following (where the curved lines indicate bonds to POLY or S, so that
the first structure, for example, represents n-butylene):

##STR00001##

[0136] As noted above, the "backbone" of the spacer group is the shortest
contiguous carbon chain linking POLY to the sulfur atom. Accordingly,
each of the structures in the second row above has a five-carbon
backbone. By this definition, moreover, the last structure shown has a
saturated backbone, although the spacer group as a whole is unsaturated.

[0137] In one exemplary polymeric reagent, depicted below, POLY is
methoxy-terminated polyethylene glycol (mPEG), Y is --(CH2)4--,
and --S--W is ortho-pyridyl disulfide (OPSS), as depicted below, or SH.
The mPEG preferably has a molecular weight in the range of 5000 to 30000
Da; e.g. about 5000, about 10000, about 20000, or about 30000 Da.

##STR00002##

[0138] In a further embodiment, the polymeric reagent is a difunctional
structure represented by W--S--Y-POLY-Y--S--W, where POLY, Y and S--W are
as defined above. Typically, though not necessarily, the polymeric
reagent is symmetrical. An exemplary polymeric reagent of this structure,
depicted below, is one in which POLY is polyethylene glycol (PEG), each Y
is --(CH2)4--, and each --S--W is ortho-pyridyl disulfide
(OPSS), as depicted below, or SH. The PEG preferably has a molecular
weight in the range of about 1000 to 5000 Da, e.g. 2000 or 3400 Da.

##STR00003##

[0139] Further exemplary polymeric reagents include reagents of the
general formula PEG-[Y--S--W]x where x is 1 or 2, Y is
--(CH2CH2CH2CH(CH3))--, and S--W is SH or
ortho-pyridyl disulfide (OPSS). When x is 1, PEG is preferably
methoxy-terminated polyethylene glycol (mPEG). Such reagents are depicted
below ("Me" represents methyl here and elsewhere):

##STR00004##

[0140] Further exemplary polymeric reagents include those of the general
formula PEG-[Y--S--W]x where x is 1 or 2, Y is
--(CH2CH2CH(CH3))--, and S--W is SH or ortho-pyridyl
disulfide (OPSS). When x is 1, PEG is preferably mPEG. Such reagents are
depicted below:

##STR00005##

[0141] Another exemplary class of polymeric reagents is that of the
general formula PEG-[Y--S--W]x where x is 1, Y is
--(CH2)4--, --S--W is SH or ortho-pyridyl disulfide (OPSS), and
PEG is terminated with the structure:

##STR00006##

[0142] Such reagents are depicted generally below:

##STR00007##

[0143] Preferably, the PEG attached to Y--SW has a molecular weight of
about 500 Da or less, or about 200 Da or less, e.g. where m=2 to 10,
preferably 2 to 4. In one embodiment, m=4. Each mPEG in the terminal
branched structure shown preferably has a molecular weight of about 5 KDa
to about 20 KDa; e.g. n=about 110 to about 450. Each mPEG may be, for
example, 5, 10, 15 or 20 KDa in molecular weight.

[0144] The reagents described herein are characterized as being
"linkerless" thiols; that is, where the water-soluble polymer is directly
linked to the hydrocarbon-based spacer group Y. For example, the oxygen
atom of a repeating alkylene glycol unit of a poly(alkylene)glycol, such
as --CH2CH2O-- in PEG, is directly linked to Y. The absence of
additional heteroatoms, particularly in linkages such as esters,
carbamates, or amides, between the active conjugating functionality,
i.e., the thiol or protected thiol, and the polymer reduces the potential
for degradation of the conjugated polymer. Moreover, the presence of such
heteroatom-containing linkages, such as amides, in such reagents, can
trigger a deleterious immune response. This potential is eliminated or
greatly reduced by the current "linkerless" reagents.

[0145] In the polymeric reagents, the polymer segment POLY preferably has
a molecular weight of at least 500 Da, particularly in embodiments where
POLY is PEG. Various preferred embodiments of the polymer segment POLY
are described in detail below. Preferably, the polymer segment "POLY" is
a polyalkylene glycol, such as a polyethylene glycol (PEG). The
polyethylene glycol may have various molecular weights, from about 88 to
about 100,000 Daltons, within the stipulations above. In selected
embodiments, the polyethylene glycol has a weight average molecular mass
from 148 (e.g. a trimer plus linking oxygen atom) to about 200 to about
40,000 Daltons. Representative molecular weights include, for example,
500, 1000, 2000, 3000, 3500, 5000, 7500, 10000, 15000, 20000, 25000,
30000, and 40000 Daltons. Generally, difunctional or polyfunctional
reagents will employ POLY or PEG segments of lower molecular weight than
monofunctional reagents.

[0146] The polymer can have a structure selected from the group consisting
of linear, branched, forked, multi-armed, and combinations thereof, as
described further herein.

[0147] The Polymer Segment, POLY

[0148] Representative water soluble polymers for use in preparing the
polymeric reagents of the invention include poly(alkylene glycols) such
as poly(ethylene glycol), poly(propylene glycol) ("PPG"), copolymers of
ethylene glycol and propylene glycol, poly(olefinic alcohol),
poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),
poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy
acid), poly(vinyl alcohol), polyphosphazene, polyoxazoline, and
poly(N-acryloylmorpholine). POLY can be a homopolymer, an alternating
copolymer, a random copolymer, a block copolymer, an alternating
tripolymer, a random tripolymer, or a block tripolymer of any of the
above. The water-soluble polymer segment is preferably a poly(ethylene
glycol) ("PEG") or a derivative thereof.

[0149] Preferably, the polymer is a hydrophilic polymer; i.e., a polymer
containing fewer than about 25 subunits of propylene oxide or other
similar hydrophobic polymer segments. The polymer may, in an alternative
embodiment, have no propylene oxide or similar hydrophobic subunits. In
one instance, the polymer is preferably not a pluronic-type polymer. In
yet another particular embodiment, the polymer is preferably not bound to
a solid phase support.

[0150] The polymer segment can have any of a number of different
geometries, for example, POLY can be linear, branched, or multiarmed.
Most typically, POLY is linear or is branched, for example, having 2
polymer arms. Although much of the discussion herein is focused upon PEG
as an illustrative POLY, the discussion and structures presented herein
can be readily extended to encompass any of the water-soluble polymer
segments described above.

[0151] Although water-soluble polymers bearing only one or two thiol
functionalities are typically used and illustrated herein, polymers
bearing two, three, four, five, six, seven, eight, nine, ten, eleven,
twelve or more such functionalities can be used. Non-limiting examples of
the upper limit of the number of thiol moieties associated with the
water-soluble polymer segment include from about 1 to about 500, from 1
to about 100, from about 1 to about 80, from about 1 to about 40, from
about 1 to about 20, and from about 1 to about 10.

[0152] A preferred type of water soluble polymer, PEG, encompasses
poly(ethylene glycol) in any of its linear, branched or multi-arm forms,
including end-capped PEG, forked PEG, branched PEG, pendant PEG, and PEG
containing one or more degradable linkages separating the monomer
subunits, to be more fully described below. The number of repeating
ethylene glycol units in a PEG polymer segment typically ranges from
about 3 to about 4,000, or from about 12 to about 3,000, or more
preferably from about 20 to about 1,000.

[0153] Preferred end-capped PEGs are those having as an end-capping moiety
such as alkoxy, substituted alkoxy, alkenyloxy, substituted alkenyloxy,
alkynyloxy, substituted alkynyloxy, aryloxy, substituted aryloxy.
Preferred end-capping groups are C1-C20 alkoxy such as methoxy,
ethoxy, and benzyloxy. The end-capping group can also advantageously
comprise a phospholipid. Exemplary phospholipids include
phosphatidylcholines, such as dilauroylphosphatidylcholine, dioleyl
phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl
phosphatidylcholine, behenoyl phosphatidylcholine, arachidoyl
phosphatidylcholine, and lecithin.

[0156] The POLY types described encompass linear polymer segments as well
as branched or multi-arm polymer segments. Examples include PEG molecules
having 2 arms, 3 arms, 4 arms, 5 arms, 6 arms, 7 arms, 8 arms or more.
Branched polymers used to prepare the thiol polymers of the invention may
possess anywhere from 2 to 300 or so reactive termini. Preferred are
branched or multi-arm PEGs having 2-8 polymer arms. Branched or multiarm
polymers for use in preparing a polymeric thiol of the invention include
those represented more generally by the formula R(POLY)n, where R is
a central or core molecule from which extends 2 or more POLY arms such as
PEG. The variable n represents the number of POLY arms, where each of the
polymer arms can independently be end-capped or possess a hydroxyl or
other reactive group at its terminus, where at least one polymer arm
possesses such a reactive group. Branched PEGs such as those represented
generally by the formula, R(PEG)n, above possess at least 2 polymer
arms, up to about 300 polymer arms (i.e., n ranges from 2 to about 300).
Preferably, such branched PEGs possess from 2 to about 25 polymer arms,
more preferably from 2 to about 20 polymer arms, and even more preferably
from 2 to about 15 polymer arms or fewer. Most preferred are multi-aimed
polymers having 3, 4, 5, 6, 7 or 8 arms.

[0157] Preferred core molecules in branched PEGs as described above are
polyols. Such polyols include aliphatic polyols having from 1 to 10
carbon atoms and from 1 to 10 hydroxyl groups, including ethylene glycol,
alkane diols, alkyl glycols, alkylidene alkyl diols, alkyl cycloalkane
diols, 1,5-decalindiol, 4,8-bis(hydroxymethyl)tricyclodecane,
cycloalkylidene diols, dihydroxyalkanes, trihydroxyalkanes,
tetrahydroxyalkanes and the like. Also, ethers of some or all of the
former class may serve as core molecules, including dipentaerythritol,
tripentaerthritol, hexaglycerol and the like. Cycloaliphatic polyols may
also be employed, including straight chained or closed-ring sugars and
sugar alcohols, such as mannitol, sorbitol, inositol, xylitol,
quebrachitol, threitol, arabitol, erythritol, adonitol, ducitol, facose,
ribose, arabinose, xylose, lyxose, rhamnose, galactose, glucose,
fructose, sorbose, mannose, pyranose, altrose, talose, tagitose,
pyranosides, sucrose, lactose, maltose, and the like. Additional
aliphatic polyols include derivatives of glyceraldehyde, glucose, ribose,
mannose, galactose, and related stereoisomers. Other core polyols that
may be used include crown ether, cyclodextrins, dextrins and other
carbohydrates such as starches and amylose. Preferred polyols include
glycerol, pentaerythritol, sorbitol, and trimethylolpropane.

[0158] A multi-arm structure corresponding to a polymeric thiol of the
invention can be represented by R--(POLY-Y--S--W)x, where POLY, Y, and
S--W are as defined above, R represents the core molecule of the multiarm
structure, and x is preferably 3 to about 8. Each of the polymer arms can
independently be end-capped or possess a thiol group at its terminus,
where at least one polymer arm possesses a thiol (or protected thiol)
group. Multi-arm PEGs suitable for preparing such structures are
available from Nektar Therapeutics (Huntsville, Ala.).

[0159] Alternatively, the polymer segment may possess an overall forked
structure, e.g., of the type PEG-(Y--S--W)2. This type of polymer
segment is useful for reaction with two active agents, where the two
active agents are positioned in a precise or predetermined distance
apart, depending upon the selection of Y.

[0160] Representative PEGs having either linear or branched structures for
use in preparing the conjugates of the invention may be purchased from
Nektar Therapeutics (Huntsville, Ala.). Illustrative structures are
described in Nektar's 2004 catalogue entitled "Polyethylene Glycol and
Derivatives for Advanced PEGylation," the contents of which are expressly
incorporated herein by reference.

[0161] In any of the representative structures provided herein, one or
more degradable linkages may be contained in the POLY segment, to allow
generation in vivo of a conjugate having a smaller POLY chain than in the
initially administered conjugate. Appropriate physiologically cleavable
linkages include but are not limited to ester, carbonate ester,
carbamate, sulfate, phosphate, acyloxyalkyl ether, acetal, and ketal.
Such linkages will preferably be stable upon storage and upon initial
administration.

[0162] The molecular weight of POLY typically falls in one or more of the
following ranges: about 100 to about 100,000 Daltons; about 500 to about
80,000 Daltons; about 1,000 to about 50,000 Daltons; about 2,000 to about
25,000 Daltons; and about 5,000 to about 20,000 Daltons. Exemplary
molecular weights include about 1,000, about 5,000, about 10,000, about
15,000, about 20,000, about 25,000, about 30,000, and about 40,000
Daltons. Low molecular weight POLYs possess molecular weights of about
250, 500, 750, 1000, 2000, or 5000 Daltons. Exemplary thiolated polymers
comprise PEGs having a molecular weight selected from the group
consisting of 5,000, 20,000, and 40,000 Daltons.

[0163] In particular embodiments of the invention, a polymeric thiol
reagent as provided herein possesses a PEG segment having one of the
following molecular weights: 500, 1000, 2000, 3000, 5000, 10,000, 15,000,
20,000, 30,000 and 40,000 Daltons.

[0164] In terms of the number of subunits, PEGs for use in the invention
will typically comprise a number of (--OCH2CH2--) subunits
falling within one or more of the following ranges: from 12 to about 4000
subunits, from about 15 to about 2000 subunits, from about 20 to about
1000 subunits, from about 25 to about 750 subunits, and from about 30 to
about 500 subunits.

[0165] Preparation of Reagents

[0166] One method of preparing "linkerless" polyalkyleneoxy-thiol reagents
is shown in Scheme I. In the reaction shown, a polyalkylene glycol having
a capping group at one terminus, such as monomethoxy PEG, is alkoxylated
with a strong base, such as NaH, and this reagent is combined with a
di(haloalkylsulfide) to form a polymeric diether disulfide. This
intermediate can then be cleaved with a reducing agent such as
dithiothreitol (DTT) to give the polyalkyleneoxy-thiol reagent.

##STR00008##

[0167] In another synthetic route, shown in Scheme 2, a
di(hydroxyalkylsulfide) is used as a core material for polymerization of
ethylene oxide, forming a PEG diether disulfide. The termini are capped
with, for example, methyl groups. The disulfide can then be cleaved with
a reducing agent such as dithiothreitol (DTT) to give the
polyalkyleneoxy-thiol reagent.

##STR00009##

[0168] A further strategy is illustrated in Scheme 3. In this route, a
reagent POLY-Y--OH, where Y is as defined above, is provided. Such
reagents can be prepared, for example, by reaction of a POLY-OH, such as
m-PEG-OH, with a strong base such as NaH to form the alkoxide salt,
followed by reaction with a haloalkanol, such as 4-bromo-1-butanol. The
terminal hydroxy group is converted to a leaving group, such as tosylate
or mesylate, and this compound is then reacted with thiourea, displacing
the leaving group. The terminal thiouronium salt is then cleaved with
base to give the terminal thiol. A variation on this scheme in which the
leaving group is a halide is employed in Example 1 below.

##STR00010##

[0169] In any of these reagents, the thiol can be protected using a thiol
protecting moiety such trityl, thioethers such as alkyl and benzyl
thioethers, including monothio, dithio and aminothio acetals, thioesters,
thiocarbonates, thiocarbamates, and sulfenyl derivatives. The use of a
protecting group during storage further reduces the tendency of the
reagents to dimerize. The thiol may also be converted to an ortho-pyridyl
disulfide (OPSS), as shown in Scheme 3, which is stable under standard
conditions of storage. Under appropriate reaction conditions, the OPSS
group reacts smoothly with thiol groups in moieties to be conjugated to
the water-soluble polymer, as shown in Example 2.

[0170] Preferably, the polymeric reagents of the invention are stored
under an inert atmosphere, such as argon or nitrogen. It is also
preferable to minimize exposure of the polymers of the invention to
moisture. Thus, preferred storage conditions are under dry argon or
another dry inert gas at temperatures below about -15° C. Storage
under low temperature conditions is preferred, since rates of undesirable
side reactions are slowed at lower temperatures. For example, when the
polymer segment is PEG, the PEG can react slowly with oxygen to form
peroxides, ultimately leading to chain cleavage and increasing the
polydispersity of the PEG reagents. In view of the above, it is
additionally preferred to store the polymers of the invention in the
dark.

[0171] Polymer Conjugates

[0172] The present invention also encompasses conjugates formed by
reaction of any of the herein described polymeric thiol reagents. In
general, the polymeric reagents of the invention are useful for
conjugation to active agents bearing at least one thiol group available
for reaction.

[0173] A conjugate of the invention will typically have the structure
POLY-[Y--S--S-A]x, where POLY is as defined above, and in preferred
embodiments is a polyethylene glycol (PEG); x is 1 to 25, and "A"
represents the residue of the active agent following conjugation. In
selected embodiments, x is 1 to 8, 1 to 6, or 1 to 4; in further
embodiments, x is 1 or x is 2. Y is a divalent linking group having at
least four carbon atoms and consisting of a saturated or unsaturated
hydrocarbon backbone which is three to ten, preferably three to eight,
carbon atoms in length and has substituents which are independently
selected from hydrogen, lower alkyl, lower alkenyl, and non-interfering
substituents as defined herein, where two such alkyl and/or alkenyl
substituents on different carbon atoms of the backbone may be linked so
as to form a cycloalkyl, cycloalkenyl, or aryl group.

[0174] In selected embodiments, Y has the structure --(CR1R2)n-,
where n is 3 to 10, preferably 3 to 8, and each of R1 and R2 is
independently selected from hydrogen, lower alkyl, lower alkenyl, and a
non-interfering substituent. Two groups R1 and R2 on different
carbon atoms may be linked to form a cycloalkyl, cycloalkenyl, or aryl
group. The sulfur atom of the thiol (or protected thiol) is attached to
an sp3 hybridized carbon atom of Y, rather than to an aryl ring or
double bond.

[0175] In selected embodiments of Y, Y is C3-C8 alkylene or a
combination of C3-C8 alkylene with C5-C8
cycloalkylene or aryl, any of which may include one or more
non-interfering substituents, as defined above. Preferably, zero to two,
more preferably zero or one, such non-interfering substituents are
included.

[0176] In further embodiments, Y is a linear or branched alkylene having
the formula --(CR1R2)n-, where n is 4 to 8, and each of R1
and R2 is independently selected from hydrogen, lower alkyl, lower
alkenyl, and a non-interfering substituent. Preferably, n is 4 to 6, and
each of R1 and R2 is independently selected from hydrogen and
methyl. In one embodiment, each of R1 and R2 is hydrogen with
respect to the n iterations of --(CR1R2)--. In another
embodiment, each of R1 and R2 is hydrogen with the exception of
R1 on a carbon adjacent said sulfur atom (a-carbon), said R1
being lower alkyl, preferably methyl or ethyl.

[0177] In embodiments of Y in which Y is --(CR1R2)n-, where n is
4 to 8, and two groups R1 and R2 on different carbon atoms are
linked to form a cycloalkyl, cycloalkenyl, or aryl group, the cycloalkyl
group is preferably a cyclopentyl or cyclohexyl group.

[0178] In another aspect, a conjugate of the invention can have the
structure POLYA-L-SS--Y-POLYB-Y'--SS-A. Each POLY is a water
soluble polymer segment, as defined above, where POLYB is of low
molecular weight, e.g. 10 KDa or less, preferably 5 KDa or less, and more
preferably 2 KDa or less, and the combined molecular weight of POLYA
and POLYB is at least 3 KDa. The molecular weight of POLYA is
generally, though not necessarily, of medium to high molecular weight,
e.g. greater than about 2 KDa, preferably 5 KDa or greater, and more
preferably 10 KDa or greater.

[0179] Each of Y and Y' is a spacer group, as defined above for Y, and
they may be the same or different. Generally, Y and Y' are identical
spacer groups. L is a linker between POLYA and the adjacent
disulfide linkage. Typically, such a linker is a direct bond or a chain
of atoms up to about ten atoms in length, containing groups preferably
selected from alkyl (C--C), alkenyl, ether, ester, amide, carbamate, and
thioester linkages. L may be, but is not necessarily, an embodiment of Y
as described herein.

[0180] These conjugates are generally the product of a reaction sequence
(illustrated in Examples 8-11 below) in which a low molecular weight
reagent of the form W--S--S--Y-POLYB--Y'--S--S--W, where S--W is a
thiol or, preferably, a thiol-reactive derivative such as OPSS, is first
reacted with a biologically active molecule A-SH, e.g. a protein having
(or modified to have) a free cysteine residue, to form an intermediate
A-S--S--Y-POLYB-Y'--S--S--W. This intermediate is then reacted with
a (typically) higher molecular weight reagent of the form
POLYA-L-S--S--W to give the final conjugate.

[0181] An advantage of such a scheme, particularly for biological
molecules with hindered thiol groups, is that a low molecular weight
reagent is able to react more efficiently with such a hindered thiol
group than would a higher molecular weight reagent. A higher molecular
weight polymer can thus be attached to A in greater yield via this scheme
than if it were reacted directly with the hindered thiol.

[0182] However, if the initial reagent W--S--S--Y-POLYB-Y'--S--S--W
lacks the hydrophilic spacer group(s) Y as described herein, the scheme
may fail due to low yields in the attachment steps. This difference is
illustrated in comparative Examples 9 and 10 below.

[0183] In general, the conjugates provided herein are preferably water
soluble or dispersible, although the polymeric thiol reagents may also be
conjugated to a solid support or surface having active thiol groups.

[0184] A thiol group, such as in a cysteine residue, for coupling to an
activated polymer of the invention may be naturally occurring (i.e.,
occurring in the protein or other molecule in its native form), or it may
be introduced, e.g. by inserting into the native sequence of a protein in
place of a naturally-occurring amino acid, using standard genetic
engineering techniques.

[0185] When the active agent contains few or only one reactive thiol
group(s), the resulting composition may advantageously contain only a
single polymer conjugate species. This is useful in conjugation to
proteins, which typically have a relatively low number of sulfhydryl
groups (as compared to other active groups such as amines) accessible for
conjugation. Covalent attachment via thiol groups can thus result in more
selective modification of the target protein. Accordingly, the use of
polymeric thiols can allow greater control over the resulting polymer
conjugate, both in the number of polymer derivatives attached to the
parent protein and the position of polymer attachment.

[0186] Candidate Molecules for Conjugation

[0187] A biologically active agent for use in preparing a conjugate of the
invention may fall into one of a number of structural classes, including
but not limited to peptides, polypeptides, proteins, antibodies,
polysaccharides, steroids, nucleotides, oligonucleotides,
polynucleotides, fats, electrolytes, small molecules (preferably
insoluble small molecules), and the like. Preferably, an active agent for
coupling to a polymer of the invention possesses a native sulfhydryl
group or is modified to contain at least one reactive sulfhydryl group
suitable for coupling.

[0189] The above exemplary biologically active agents are meant to
encompass, where applicable, analogues, agonists, antagonists,
inhibitors, isomers, and pharmaceutically acceptable salt forms thereof.
In reference to peptides and proteins, the invention is intended to
encompass synthetic, recombinant, native, glycosylated, and
non-glycosylated forms, as well as biologically active fragments thereof.
The above biologically active proteins are additionally meant to
encompass variants having one or more amino acids substituted (e.g.,
cysteine), deleted, or the like, as long as the resulting variant protein
possesses at least a certain degree of activity of the parent (native)
protein.

[0194] Suitable conjugation conditions are those conditions of time,
temperature, pH, reagent concentration, solvent, and the like sufficient
to effect conjugation between a polymeric thiol reagent and an active
agent. As is known in the art, the specific conditions depend upon, among
other things, the active agent, the type of conjugation desired, the
presence of other materials in the reaction mixture, and so forth.
Sufficient conditions for effecting conjugation in any particular case
can be determined by one of ordinary skill in the art upon a reading of
the disclosure herein, reference to the relevant literature, and/or
through routine experimentation.

[0195] Exemplary conjugation conditions include carrying out the
conjugation reaction at a pH of from about 6 to about 10, and at, for
example, a pH of about 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10. The
reaction is allowed to proceed from about 5 minutes to about 72 hours,
preferably from about 30 minutes to about 48 hours, and more preferably
from about 4 hours to about 24 hours or less. Temperatures for
conjugation reactions are typically, although not necessarily, in the
range of from about 0° C. to about 40° C.; conjugation is
often carried out at room temperature or less. Conjugation reactions are
often carried out in a buffer such as a phosphate or acetate buffer or
similar system.

[0196] With respect to reagent concentration, an excess of the polymeric
reagent is typically combined with the active agent. In some cases,
however, it is preferred to have stoichiometric amounts of the number of
reactive groups on the polymeric reagent to the amount of active agent.
Exemplary ratios of polymeric reagent to active agent include molar
ratios of about 1:1 (polymeric reagent:active agent), 1.5:1, 2:1, 3:1,
4:1, 5:1, 6:1, 8:1, or 10:1. The conjugation reaction is allowed to
proceed until substantially no further conjugation occurs, which can
generally be determined by monitoring the progress of the reaction over
time.

[0197] Progress of the reaction can be monitored by withdrawing aliquots
from the reaction mixture at various time points and analyzing the
reaction mixture by SDS-PAGE or MALDI-TOF mass spectrometry or any other
suitable analytical method. Once a plateau is reached with respect to the
amount of conjugate formed or the amount of unconjugated polymer
remaining, the reaction is assumed to be complete. Typically, the
conjugation reaction takes anywhere from minutes to several hours (e.g.,
from 5 minutes to 24 hours or more). The resulting product mixture is
preferably, but not necessarily purified, to separate out excess
reagents, unconjugated reactants (e.g., active agent) undesired
multi-conjugated species, and free or unreacted polymer. The resulting
conjugates can then be further characterized using analytical methods
such as MALDI, capillary electrophoresis, gel electrophoresis, and/or
chromatography.

[0198] More preferably, a polymeric thiol of the invention is typically
conjugated to a sulfhydryl-containing active agent at a pH of about 6-9
(e.g., at 6, 6.5, 7, 7.5, 8, 8.5, or 9), more preferably at a pH of about
7-9, and even more preferably at a pH of about 7 to 8. Generally, a
slight molar excess of polymeric reagent is employed, for example, a 1.5
to 15-fold molar excess, preferably a 2-fold to 10 fold molar excess.
Reaction times generally range from about 15 minutes to several hours,
e.g., 8 or more hours, at room temperature. For sterically hindered
sulfhydryl groups, required reaction times may be significantly longer.

[0199] Purification of Conjugates

[0200] Optionally, conjugates produced by reacting a polymeric thiol of
the invention with a biologically active agent are purified to
obtain/isolate different species, e.g., PEG-species, or to remove
undesirable reaction side-products.

[0201] If desired, PEG conjugates having different molecular weights can
be isolated using gel filtration chromatography. While this approach can
be used to separate PEG conjugates having different molecular weights,
this approach is generally ineffective for separating positional isomers
having different PEGylation sites within a protein. For example, gel
filtration chromatography can be used to separate from each other
mixtures of PEG 1-mers, 2-mers, 3-mers, etc., although each of the
recovered PEG-mer compositions may contain PEGs attached to different
reactive groups within the protein.

[0202] Gel filtration columns suitable for carrying out this type of
separation include Superdex® and Sephadex® columns available from
Amersham Biosciences. Selection of a particular column will depend upon
the desired fractionation range desired. Elution is generally carried out
using a non-amine based buffer, such as phosphate, acetate, or the like.
The collected fractions may be analyzed by a number of different methods,
for example, (i) OD at 280 nm for protein content, (ii) BSA protein
analysis, (iii) iodine testing for PEG content (Sims, G. E. C. et al.,
Anal. Biochem, 107, 60-63, 1980), or alternatively, (iv) by running an
SDS PAGE gel, followed by staining with barium iodide.

[0203] Separation of positional isomers can be carried out by reverse
phase chromatography using, for example, an RP-HPLC C18 column (Amersham
Biosciences or Vydac) or by ion exchange chromatography using an ion
exchange column, e.g., a Sepharose® ion exchange column available from
Amersham Biosciences. Either approach can be used to separate
PEG-biomolecule isomers having the same molecular weight (positional
isomers).

[0204] Depending upon the intended use for the resulting PEG-conjugates,
following conjugation, and optionally additional separation steps, the
conjugate mixture may be concentrated, sterile filtered, and stored at
low temperatures from about -20° C. to about -80° C.
Alternatively, the conjugate may be lyophilized, either with or without
residual buffer and stored as a lyophilized powder. In some instances, it
is preferable to exchange a buffer used for conjugation, such as sodium
acetate, for a volatile buffer such as ammonium carbonate or ammonium
acetate, that can be readily removed during lyophilization, so that the
lyophilized protein conjugate powder formulation is absent residual
buffer. Alternatively, a buffer exchange step may be used using a
formulation buffer, so that the lyophilized conjugate is in a form
suitable for reconstitution into a formulation buffer and ultimately for
administration to a mammal.

[0205] Pharmaceutical Compositions

[0206] The present invention also includes pharmaceutical preparations
comprising a conjugate as provided herein in combination with a
pharmaceutical excipient. Generally, the conjugate itself will be in a
solid form (e.g., a precipitate or a lyphilizate) or in solution, which
can be combined with a suitable pharmaceutical excipient that can be in
either solid or liquid form.

[0208] A carbohydrate such as a sugar, a derivatized sugar such as an
alditol, aldonic acid, an esterified sugar, and/or a sugar polymer may be
present as an excipient. Specific carbohydrate excipients include, for
example: monosaccharides, such as fructose, maltose, galactose, glucose,
D-mannose, sorbose, and the like; disaccharides, such as lactose,
sucrose, trehalose, cellobiose, and the like; polysaccharides, such as
raffinose, melezitose, maltodextrins, dextrans, starches, and the like;
and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol,
sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.

[0211] An antioxidant can be present in the preparation as well.
Antioxidants are used to prevent oxidation, thereby preventing the
deterioration of the conjugate or other components of the preparation.
Suitable antioxidants for use in the present invention include, for
example, ascorbyl palmitate, butylated hydroxyanisole, butylated
hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate,
sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite,
and combinations thereof.

[0212] A surfactant may be present as an excipient. Exemplary surfactants
include: polysorbates, such as "Tween 20" and "Tween 80," and pluronics
such as F68 and F88 (both of which are available from BASF, Mount Olive,
N.J.); sorbitan esters; lipids, such as phospholipids such as lecithin
and other phosphatidylcholines, phosphatidylethanolamines (although
preferably not in liposomal form), fatty acids and fatty esters;
steroids, such as cholesterol; and chelating agents, such as EDTA, zinc
and other such suitable cations.

[0214] The pharmaceutical preparations encompass all types of formulations
and in particular those that are suited for injection, e.g., powders that
can be reconstituted as well as suspensions and solutions. The amount of
the conjugate (i.e., the conjugate formed between the active agent and
the polymer described herein) in the composition will vary depending on a
number of factors, but will optimally be a therapeutically effective dose
when the composition is stored in a unit dose container (e.g., a vial).
In addition, the pharmaceutical preparation can be housed in a syringe. A
therapeutically effective dose can be determined experimentally by
repeated administration of increasing amounts of the conjugate in order
to determine which amount produces a clinically desired endpoint.

[0215] The amount of any individual excipient in the composition will vary
depending on the activity of the excipient and particular needs of the
composition. Typically, the optimal amount of any individual excipient is
determined through routine experimentation, i.e., by preparing
compositions containing varying amounts of the excipient (ranging from
low to high), examining the stability and other parameters, and then
determining the range at which optimal performance is attained with no
significant adverse effects.

[0216] Generally, however, the excipient will be present in the
composition in an amount of about 1% to about 99% by weight, preferably
from about 5%-98% by weight, more preferably from about 15-95% by weight
of the excipient, with concentrations less than 30% by weight most
preferred.

[0218] The pharmaceutical preparations of the present invention are
typically, although not necessarily, administered via injection and are
therefore generally liquid solutions or suspensions immediately prior to
administration. The pharmaceutical preparation can also take other forms
such as syrups, creams, ointments, tablets, powders, and the like. Other
modes of administration are also included, such as pulmonary, rectal,
transdermal, transmucosal, oral, intrathecal, subcutaneous,
intra-arterial, and so forth.

[0219] As previously described, the conjugates can be administered
injected parenterally by intravenous injection, or less preferably by
intramuscular or by subcutaneous injection. Suitable formulation types
for parenteral administration include ready-for-injection solutions, dry
powders for combination with a solvent prior to use, suspensions ready
for injection, dry insoluble compositions for combination with a vehicle
prior to use, and emulsions and liquid concentrates for dilution prior to
administration, among others.

[0220] Methods of Administration

[0221] The invention also provides a method for administering a conjugate
as provided herein to a patient suffering from a condition that is
responsive to treatment with conjugate. The method comprises
administering, generally via injection, a therapeutically effective
amount of the conjugate (preferably provided as part of a pharmaceutical
preparation). The method of administering may be used to treat any
condition that can be remedied or prevented by administration of the
particular conjugate. Those of ordinary skill in the art appreciate which
conditions a specific conjugate can effectively treat. The actual dose to
be administered will vary depend upon the age, weight, and general
condition of the subject as well as the severity of the condition being
treated, the judgment of the health care professional, and conjugate
being administered. Therapeutically effective amounts are known to those
skilled in the art and/or are described in the pertinent reference texts
and literature. Generally, a therapeutically effective amount will range
from about 0.001 mg to 100 mg, preferably in doses from 0.01 mg/day to 75
mg/day, and more preferably in doses from 0.10 mg/day to 50 mg/day.

[0222] The unit dosage of any given conjugate (again, preferably provided
as part of a pharmaceutical preparation) can be administered in a variety
of dosing schedules depending on the judgment of the clinician, needs of
the patient, and so forth. The specific dosing schedule will be known by
those of ordinary skill in the art or can be determined experimentally
using routine methods. Exemplary dosing schedules include, without
limitation, administration five times a day, four times a day, three
times a day, twice daily, once daily, three times weekly, twice weekly,
once weekly, twice monthly, once monthly, and any combination thereof.
Once the clinical endpoint has been achieved, dosing of the composition
is halted.

[0223] Cleavage of the water-soluble polymer portion of the conjugate in
vivo, when desired, can be effected through the use of physiologically
cleavable and/or enzymatically degradable linkages, such as urethane,
amide, carbonate or ester-containing linkages, in the polymer backbone.
In this way, clearance of the conjugate (via cleavage of water-soluble
polymer portions) can be modulated by selecting the polymer molecular
size and the type functional group that would provide the desired
clearance properties. One of ordinary skill in the art can determine the
proper molecular size of the polymer as well as the cleavable linkages.
For example, one of ordinary skill in the art, using routine
experimentation, can determine a proper molecular size and cleavable
functional group by first preparing a variety of polymer derivatives with
different polymer weights and cleavable linkages, and then obtaining the
clearance profile (e.g., through periodic blood or urine sampling) by
administering the polymer derivative to a patient and taking periodic
blood and/or urine sampling. Once a series of clearance profiles have
been obtained for each tested conjugate, a suitable conjugate can be
identified.

[0224] All articles, books, patents, patent publications and other
publications referenced herein are incorporated by reference in their
entireties.

EXAMPLES

[0225] The following examples illustrate but in no way are intended to
limit the scope of the present invention. In one aspect, the Examples
illustrate the increased stability, during synthesis and conjugation, of
the polymeric thiol reagents of the invention.

[0226]1H NMR data was obtained using a 400 MHz spectrometer
manufactured by Bruker.

[0227] PEG reagents referred to are available from Nektar Therapeutics,
Huntsville, Ala.

[0230] Iodometric analysis showed that the product contained 94% thiol
groups. The NMR data, above, indicated that the product contained a very
small amount (1.2 mol % by NMR) of disulfide-linked dimer, formed by
oxidation of thiol groups. No further purification of the thiol was
required.

[0231] In contrast, the analogous preparation of mPEG5000-ethanethiol
(i.e. the corresponding reagent containing only a two-carbon spacer
between the PEG and the thiol group) from mPEG5000-mesylate and
thiourea, conducted in a similar manner, produced product containing
about 15 mol % of disulfide-linked dimer containing dithiol group (see
e.g. WO 2004/063250). This level of dimer necessitates further
purification or additional chemical treatment to convert the dimer to the
desired PEG-thiol.)

[0237] To a solution of PEG2000-di(butyl bromide) (10.0 g, 0.0100
equiv.) in anhydrous ethyl alcohol (100 ml), thiourea (7.68 g, 99%, 0.100
mol) was added, and the mixture was stirred overnight at 78° C.
under argon. The solvent was removed by distillation under reduced
pressure, and the residue was dissolved in 3.3% aqueous NaOH (180 ml).
This solution was heated for 2.5 h at 85° C. under argon. After
cooling the solution to 35° C., 60 ml deionized water was added,
and the pH was adjusted to 3 with 10% phosphoric acid. The solution was
washed with 50 ml ethyl acetate, and the product was extracted with
dichloromethane. The extract was dried with anhydrous sodium sulfate and
the solvent was removed by distillation under reduced pressure. The crude
product was recrystallised from isopropyl alcohol and dried under vacuum.
Yield 7.8 g. NMR (CDCl3): 1.35 ppm (t, --CH2--SH, 1H), 1.69 ppm
(m, --O--CH2--CH2-CH2--CH2--SH, 4H), 2.55 ppm (m,
--CH2--SH, 2H), 2.69 ppm (t, --CH2--S--S--CH2--, 4H, 1.9
mol %), 3.64 ppm (s, PEG backbone).

[0238] The NMR data, above, indicated that the product contained a
relatively small amount (1.9 mol % by NMR) of disulfide-linked dimer,
formed by oxidation of thiol groups. No further purification of the thiol
was required.

[0239] In contrast, the analogous preparation of
PEG2000-di-ethanethiol (i.e. the corresponding reagent containing
only a two-carbon spacer between the PEG and the thiol group) from
PEG2000-di-mesylate and thiourea, conducted in a similar manner,
produced product containing about 41 mol % of disulfide-linked dimer
containing dithiol group.

[0245] To a solution of mPEG10,000-butyl bromide (10.0 g, 0.0010 mol)
in anhydrous ethyl alcohol (100 ml), thiourea (0.77 g, 99%, 0.0100 mol)
was added, and the mixture was stirred overnight at 78° C. under
argon. The solvent was removed by distillation under reduced pressure,
and the residue was dissolved in 1.0% aqueous NaOH (90 ml). This solution
was heated for 3 h at 85° C. under argon. After cooling the
solution to room temperature NaCl (10 g) was added and the pH was
adjusted to 3 with 10% phosphoric acid. The product was extracted with
dichloromethane. The extract was dried with anhydrous sodium sulfate, and
the solvent was removed by distillation under reduced pressure. The crude
product was dissolved in small amount of dichloromethane, precipitated
with ethyl ether and dried under vacuum. Yield 9.0 g. NMR (CDCl3):
1.35 ppm (t, --CH2--SH, 1H), 1.69 ppm (m,
--O--CH2--CH2--CH2--CH2--SH, 4H), 2.55 ppm (m,
--CH2--SH, 2H), 2.69 ppm (t, --CH2--S--S--CH2--, 4H, 4.8
mol %), 3.38 ppm (s, --OCH3, 3H), 3.64 ppm (s, PEG backbone). The
NMR data, above, indicated that the product contained a relatively small
amount (4.8 mol % by NMR) of disulfide-linked dimer, formed by oxidation
of thiol groups. No further purification of the thiol was required.

[0252] To a solution of mPEG20,000-butyl bromide (10.0 g, 0.5 mmol)
in anhydrous ethyl alcohol (100 ml), thiourea (0.39 g, 99%, 0.0051 mol)
was added, and the mixture was stirred overnight at 78° C. under
argon. The solvent was removed by distillation under reduced pressure,
and the residue was dissolved in 1.0% aqueous NaOH (90 ml). This solution
was heated for 3 h at 85° C. under argon. After cooling the
solution to room temperature NaCl (10 g) was added, and the pH was
adjusted to 3 with 10% phosphoric acid. The product was extracted with
dichloromethane. The extract was dried with anhydrous sodium sulfate, and
the solvent was removed by distillation under reduced pressure. The crude
product was dissolved in a small amount of dichloromethane, precipitated
with ethyl ether and dried under vacuum. Yield 8.2 g. NMR (CDCl3):
1.35 ppm (t, --CH2--SH, 1H), 1.69 ppm (m,
--O--CH2--CH2-CH2--CH2--SH, 4H), 2.55 ppm (m,
--CH2--SH, 2H), 2.69 ppm (t, --CH2--S--S--CH2--, 4H, 3.4
mol %), 3.38 ppm (s, --OCH3, 3H), 3.64 ppm (s, PEG backbone). The
NMR data, above, indicated that the product contained a relatively small
amount (3.4 mol % by NMR) of disulfide-linked dimer, formed by oxidation
of thiol groups. No further purification of the thiol was required.

[0258] To a solution of mPEG30,000-butyl bromide (10.0 g, 0.00033
mol) in anhydrous ethyl alcohol (100 ml), thiourea (0.26 g, 99%, 0.00338
mol) was added, and the mixture was stirred overnight at 78° C.
under argon. The solvent was removed by distillation under reduced
pressure, and the residue was dissolved in 1.0% aqueous NaOH (90 ml).
This solution was heated for 2.5 h at 85° C. under argon. After
cooling to room temperature NaCl (10 g) was added and the pH was adjusted
to 3 with 10% phosphoric acid. The product was extracted with
dichloromethane. The extract was dried with anhydrous sodium sulfate and
the solvent was removed by distillation under reduced pressure. The crude
product was dissolved in a small amount of dichloromethane, precipitated
with ethyl ether and dried under vacuum. Yield 9.4 g. NMR (CDCl3):
1.35 ppm (t, --CH2--SH, 1H), 1.69 ppm (m,
--O--CH2--CH2--CH2--CH2--SH, 4H), 2.55 ppm (m,
--CH2--SH, 2H), 2.69 ppm (t, --CH2--S--S--CH2--, 4H, 3.8
mol %), 3.38 ppm (s, --OCH3, 3H), 3.64 ppm (s, PEG backbone). The
NMR data, above, indicated that the product contained a relatively small
amount (3.8 mol % by NMR) of disulfide-linked dimer, formed by oxidation
of thiol groups. No further purification of the thiol was required.

Conjugation of BSA with mPEG5000-4C-OPSS and with mPEG5000-MAL
(MAL=maleimide) (Comparative)

[0261] Reduction of BSA (Cleavage of Disulfide Linkages)

[0262] A 3.1 mg sample of BSA was added to a 5 mL ReactiVial®
containing 3.1 mL 1×PBS pH 7.5. The solution was placed on a stir
plate at medium speed. A 4.62 mg sample of dithiothreitol (DTT) was added
to the solution with stirring and allowed to react for 2 hrs at room
temperature, reducing the sample completely.

[0263] The reaction mixture was placed in a 350 mL Amicon StirCell with a
10,000 MW PES membrane for removal of DTT. Buffer (1×PBS pH 7.5)
was added to a volume of 350 mL, with stirring to prevent settling.
Pressure was applied to the apparatus (60 psi) until the volume was
reduced to <10 mL. PBS was again added to a volume of 350 mL, and the
process was repeated twice. A 1 mL aliquot was frozen for standards
(gels, HPLC, etc.), and the remaining volume was used in the conjugation
step.

[0264] Conjugation

[0265] Reduced BSA from step A (4 mL) was combined with 2.35 mg (10×
excess) mPEG5K-4C-OPSS, described in Example 1, in a 5 mL ReactiVial
on a stir plate set to the medium setting. A similar reaction mixture was
prepared using reduced BSA from step A (4 mL) and 2.35 mg (10×
excess) mPEG5K-MAL. (In mPEG-MAL, available from Nektar
Therapeutics, Huntsville, Ala., maleimide is attached via the ring
nitrogen to the terminal --OCH2CH2-- of mPEG.) The vials were
left at room temperature for 60 hrs.

[0266] Analysis

[0267] The reaction mixtures were run on 10% Bis-Tris NuPAGE Gels
(Invitrogen) using the following conditions.

[0270] The PEG5K-MAL-BSA conjugation reaction yielded 39.8% mono
PEGmers (58440 MW bands), and the mPEGSK-4C-OPSS-BSA conjugation
reaction yielded 42.2% mono PEGmers. Accordingly, the conjugation
behavior of the polymeric thiol reagent of the invention was better than
that observed for a reference polymeric reagent (maleimide-terminated
polymer), indicating that significant dimerization of PEG-OPSS, which is
typical for the corresponding reagent based on mPEG-ethanethiol, did not
occur.

[0272] A fifty-fold excess (relative to the amount of G-CSF in a measured
aliquot of stock G-CSF solution) of
mPEG10,000-(CH2)4-orthopyridyl disulfide
(mPEG10,000-4C-OPSS), as prepared in Example 3, was dissolved in
dimethylsulfoxide (DMSO) to form a 10% reagent solution. The 10% reagent
solution was quickly added to the aliquot of stock G-CSF solution (0.4
mg/ml in sodium phosphate buffer, pH 7.0) and mixed well. To allow for
coupling of the mPEG-OPSS reagent to the free (i.e.,
nonintraprotein-disulfide bond participating) cysteine residue at
position 17 of G-CSF, the reaction solution was placed on a RotoMix (Type
48200, Thermolyne, Dubuque Iowa) to facilitate conjugation at 37°
C. After thirty minutes, another fifty-fold excess of
mPEG10,000-4C-OPSS was added to the reaction solution, followed by
mixing first for thirty minutes at 37° C., and then for two hours
at room temperature, to thereby form an mPEG10,000-G-CSF conjugate
solution.

[0273] The mPEG10,000-G-CSF conjugate solution was characterized by
SDS-PAGE and RP-HPLC. The PEGylation reaction was determined to yield 36%
of mPEG10,000-G-CSF conjugate (a monoPEGylated conjugate at a
cysteine residue of G-CSF). Cation-exchange chromatography was used to
purify the conjugate.

[0274] The same approach can be used to prepare other conjugates using
mPEG-4C--OPSS reagents having other molecular weights.

[0275] Examples 8-10, following, employ an approach (illustrated
schematically below) in which a polymeric reagent having a relatively low
molecular weight (PEGB in the schematic) is initially attached to a
moiety to be conjugated (A), followed by attachment of a higher molecular
weight polymeric reagent (PEGA in the schematic) to the polymeric
portion of the conjugate formed from attachment of the low molecular
weight reagent to the conjugated moiety. Using this approach, it is
possible to efficiently modify a hindered site. In the Examples below,
the hindered site is the partially buried free thiol-containing cysteine
residue of G-CSF.

##STR00013##

Example 8a

PEGylation of G-CSF with PEG2000-di-((CH2)4-orthopyridyl
disulfide) and mPEG20,000-butanethiol

##STR00014##

[0277] In this Example, the bifunctional PEG-di-(4C-OPSS) reagent is
inserted into the sterically hindered free thiol via a disulfide linkage,
followed by the coupling of mPEG20K-butanethiol to the free residue
of the PEG2,000-di-(4C-OPSS) reagent, via a further disulfide
linkage.

[0278] PEG2,000-di-(4C-OPSS), as prepared in Example 2, stored at
-20° C. under argon, was warmed to ambient temperature. A
fifty-fold excess (relative to the amount of G-CSF in a measured aliquot
of stock G-CSF solution) of the reagent was dissolved in DMSO to form a
10% solution. The 10% reagent solution was quickly added to the aliquot
of stock G-CSF solution (0.4 mg/ml in sodium phosphate buffer, pH 7.0)
and mixed well. The reaction solution was placed on a RotoMix (Type
48200, Thermolyne, Dubuque Iowa), and was allowed to mix for one hour at
37° C., and then for two hours at room temperature. After the
reaction was complete, the reaction solution was dialyzed against sodium
phosphate buffer, pH 7.0, to remove excess free
PEG2,000-di-(4C-OPSS).

[0279] A fifty-fold excess (relative to G-CSF) of
mPEG20,000-butanethiol, as prepared in Example 4B, was then added to
the dialyzed solution of intermediate conjugate, followed by mixing for
one hour at room temperature and then overnight at 4° C., to
thereby form the mPEG20,000-PEG2,000-GCSF conjugate. The
product was characterized by SDS-PAGE and RP-HPLC.

[0280] This approach can be used to prepare other conjugates, using
PEG-di-(4C-OPSS) and mPEG-4C--SH having other molecular weights, again
where the PEG-di-(4C-OPSS) reagent is preferably of relatively low
molecular weight.

Example 8b

PEGylation of G-CSF with PEG2000-di-((CH2)4-orthopyridyl
disulfide) and mPEG30,000-butanethiol

[0281] The procedure of Example 8a was repeated using corresponding
amounts of PEG2000-di-((CH2)4-orthopyridyl disulfide) and
mPEG30,000-butanethiol, to obtain the corresponding
mPEG30,000-PEG2,000-GCSF conjugate.

[0282] Other conjugates can be similarly prepared using PEG-di-(4C-OPSS)
and mPEG-4C--SH having other molecular weights.

[0283] Examples 9-10 below differ from each other in that the low
molecular weight PEG species of Example 9 contains a four-carbon
hydrophilic linker of the invention, while that of Example 10 contains
only a two-carbon linker. It can be seen that the linker of the invention
provides significantly greater yields of conjugate.

Example 9

PEGylation of G-CSF with PEG2000-di-((CH2)4-orthopyridyl
disulfide) and branched PEG240,000-thiol

##STR00015##

[0285] Again, these examples employ an approach involving initial
attachment of a polymeric reagent having a relatively small molecular
weight (in this Example, PEG2,000-di-(4C--OPSS)) to a G-CSF moiety,
followed by attachment of a relatively large molecular weight polymeric
reagent (in this Example, branched PEG240,000-thiol) to residue of
the PEG2,000-di-(4C--OPSS) reagent, through another disulfide
linkage.

[0286] PEG2,000-di-(4C-OPSS) as prepared in Example 2, stored at
-20° C. under argon, was warmed to ambient temperature. A
fifty-fold excess (relative to the amount of G-CSF in a measured aliquot
of stock G-CSF solution) of the warmed PEG2,000-di-(4C-OPSS) was
dissolved in DMSO to form a 10% reagent solution. The 10% reagent
solution was quickly added to the aliquot of stock G-CSF solution (0.4
mg/ml in sodium phosphate buffer, pH 7.0) and mixed well. The solution
was placed on a RotoMix (Type 48200, Thermolyne, Dubuque Iowa) and
allowed to mix for one hour at 37° C., then for two hours at room
temperature. After the reaction was complete, the reaction solution was
dialyzed against sodium phosphate buffer, pH 7.0, to remove excess
PEG2,000-di-(4C-OPSS).

[0287] A seventy five-fold excess (relative to G-CSF) of
PEG240,000-thiol (Nektar Therapeutics) was then added to the
dialyzed conjugate solution, followed by mixing for three hours at room
temperature and then overnight at 4° C., to form a
PEG240,000-PEG2,000-G-CSF conjugate. The conjugate was
characterized by SDS-PAGE and RP-HPLC. The final yield of conjugate
obtained was 35%.

Example 10

Comparative

PEGylation Reaction of G-CSF with PEG2000-di-((CH2)-orthopyridyl
disulfide) and PEG240,000-thiol

##STR00016##

[0289] The reaction procedure of Example 10 was essentially duplicated,
using a low molecular weight PEG thiol reagent having a two-carbon rather
than a four-carbon linker.

[0290] Accordingly, PEG2,000-di-(2C--OPSS) from Nektar Therapeutics,
stored at -20° C. under argon, was warmed to ambient temperature.
A fifty-fold excess (relative to the amount of G-CSF in a measured
aliquot of stock G-CSF solution) of the reagent was dissolved in DMSO to
form a 10% solution. This solution was quickly added to the aliquot of
stock G-CSF solution (0.4 mg/ml in sodium phosphate buffer, pH 7.0) and
mixed well. The reaction solution was placed on a RotoMix (Type 48200,
Thermolyne, Dubuque Iowa) and was allowed to mix for one hour at
37° C., then for two hours at room temperature. After the reaction
was complete, the reaction solution was dialyzed against sodium phosphate
buffer, pH 7.0, to remove excess PEG2,000-di-(2C--OPSS).

[0291] A seventy-fold excess (relative to G-CSF) of branched
PEG240,000-thiol (Nektar Therapeutics) was added to the dialyzed
conjugate solution, followed by mixing for three hour at room temperature
and overnight at 4° C. However, SDS-PAGE and RP-HPLC analysis
showed no detectable amount of the desired
PEG240,000-PEG2,000-G-CSF conjugate.

[0292] Evidence suggests that the ethylene (C2)-linked PEG-OPSS reagent
undergoes reductive cleavage to effectively destroy the reagent before it
reacts with the target protein. The butylene (C4)-linked reagent is more
stable to such cleavage and thereby survives to give a much higher yield
of conjugate.

[0298] To a solution of 1-methyl-4-bromo-1-butanol (9.0 g, 0.05384 mol)
and benzyl 2,2,2-trichloroacetimidate (16.3 g,) in a mixture of anhydrous
cyclohexane (100 ml) and anhydrous dichloromethane (50 ml) cooled to
0° C., trifluoromethanesulfonic acid (1.0 ml) was added, and the
mixture was stirred overnight at room temperature under argon. The
mixture was filtered, washed with a saturated solution of NaHCO3
(250 ml) and deionized water (250 ml), and dried with anhydrous
Na2SO4. The solvents were removed by distillation under reduced
pressure. The crude product (9.2 g) was subjected to vacuum distillation,
giving 7.2 g of colorless viscous liquid.

[0301] To an azeotropically dried solution of mPEG5000 (20.0 g, 0.004
mole) (NOF Corporation) in anhydrous toluene (200 ml), a 1.0M solution of
potassium tert-butoxide in tert-butanol (16.0 ml, 0.0160 mole) and
1-bromo-4-methyl-4-benzyloxybutane (3.10 g, 0.012 mole) were added. The
reaction mixture was stirred for 20 hours at 70° C. under
nitrogen. The resulting mixture was filtered and concentrated under
vacuum to dryness. The crude product was dissolved in 30 ml of
dichloromethane and precipitated with 500 ml of isopropanol at
0-5° C. The final product was collected through vacuum filtration
and dried under vacuum overnight. Yield: 17.4 g.

[0304] A mixture of mPEG5000-4-methyl-4-benzyloxybutane (15.0 g,
0.00300 mole), ethyl alcohol (150 ml), and palladium (10% on active
carbon, 1.5 g) was hydrogenated overnight under 45 psi of hydrogen. The
mixture was filtered and the solvent was removed by distillation under
reduced pressure. The crude product was dissolved in dichloromethane (25
ml) and precipitated with 400 ml isopropyl alcohol at 0-5° C. The
product was filtered off and dried under reduced pressure. Yield: 13.1 g.

[0306] A solution of mPEG5000-4-methyl-4-butanol (10.0 g, 0.0020
mole) in toluene (100 ml) was azeotropically dried by distilling off
toluene under reduced pressure. The dried
mPEG5000-4-methyl-4-butanol was dissolved in a mixture of anhydrous
toluene (100 ml) and anhydrous dichloromethane (20 ml). Triethylamine
(0.9 ml, 0.0030 mole) and methanesulfonyl chloride (0.45 ml, 0.0026 mole)
were added, and the mixture was stirred overnight at room temperature
under nitrogen. The solvents were removed by distillation under reduced
pressure. The residue was dissolved in dichloromethane (15 ml), and 250
ml isopropyl alcohol was added. The precipitated product was filtered and
dried under vacuum to yield 8.9 g of the white solid powder.

[0309] To a solution of mPEG5000-4-methyl-4-methanesulfonylbutane
(8.0 g, 0.0016 mol) in anhydrous ethyl alcohol (80 ml), thiourea (1.24 g,
0.0163 mol) was added, and the mixture was stirred overnight at
78° C. under argon. The solvent was removed by distillation under
reduced pressure, and the residue was dissolved in 1% aqueous NaOH (84
ml). This solution was heated for 2.5 h at 85° C. under argon.
After cooling the solution to 35° C., the pH was adjusted to 3
with 10% phosphoric acid. NaCl (24 g) was added, and the product was
extracted with dichloromethane. The extract was dried with anhydrous
sodium sulfate, and the product was precipitated with cold ethyl ether.
Yield 7.3 g.

[0311] The NMR data, above, indicated that the product contained a very
small amount (0.7 mol % by NMR) of disulfide-linked dimer, formed by
oxidation of thiol groups. No further purification of the thiol was
required.

mPEG5000CH2--CH2--CH2--CH(CH3)--OPSS

##STR00023##

[0313] To a solution of mPEG5000-4-methyl-4-butanethiol (2.0 g,
0.0004 mol) in anhydrous methyl alcohol (40 ml), 2,2'-dipyridyl disulfide
(0.18 g, 0.00082 mol) was added, and the mixture was stirred for 4 h at
room temperature under argon. The solvent was removed by distillation
under reduced pressure, the residue was dissolved in dichloromethane (5
ml), and the product was precipitated with 50 ml of cold ethyl ether.
Yield 1.7 g.